正常成人胰岛移植治疗糖尿病的实验研究

目的探讨正常成人胰岛移植治疗糖尿病的作用。方法 2012年1月至2013年1月健康成人胰腺供体共13例。获取胰腺后经胶原酶消化并进行胰岛提取纯化,将纯化后的胰岛移植到糖尿病裸鼠肾包膜下为实验组,生理盐水代替胰岛移植为对照组,各13只。术后通过检测糖尿病裸鼠的血糖变化和组织学检查来判定胰岛移植后的存活情况和纠正血糖的效果。结果成人胰腺消化后胰岛产量为纯化前（3608±403）IEQ/g,纯化后（2820±318）IEQ/g,纯度为85%。实验组84.6%（11/13）糖尿病裸鼠在移植术后第1天,血糖降至正常,对照组血糖无明显变化。两组糖尿病裸鼠存活时间：实验组存活时间35～57d,中位存活时间46d。对照组存活时间5～12d,中位存活时间7d。两组比较差异具有统计学意义,Log-rank（Mantel-Cox）值为8.74,P值为0.0012。结论成人胰腺消化后,可得到高纯度活力正常的胰岛,并能纠正糖尿病裸鼠的高血糖。正常成人胰岛移植可能成为临床糖尿病患者的一种有效治疗方法。     

国产胶原酶在小鼠胰岛分离中的效果研究#br#

目的 研究一种国产胶原酶在小鼠胰岛分离中的分离效果,探索该胶原酶在胰岛分离中应用的 可行性。方法 将国产胶原酶分别配成不同浓度的胶原酶溶液和含中性蛋白酶的胶原酶的溶液,经胰管逆行灌注胶原酶的方法进行小鼠胰岛分离,采用Ficoll液对胰岛进行纯化,计数所得的胰岛细胞团,培养 6 h后检测活性,对最佳分离结果的胰岛做同系糖尿病小鼠的肾被膜下移植,监测术后血糖和进行组织学 检查,以进口Sigma胶原酶V为对照。结果 国产胶原酶在小鼠胰腺消化时间、所得胰岛数量和活性效果 上跟进口胶原酶V有一定差距（P＜0.01）,但含中性蛋白酶的国产胶原酶组在小鼠胰岛分离数量和当量上 与进口胶原酶组无差异（P＞0.05）,分离的胰岛在体内移植后降血糖效果与进口胶原酶组无差异（P＞0.05）。 结论 国产胶原酶结合中性蛋白酶可用于小鼠胰岛的分离。     

The ratio between class II and class I collagenase determines the amount of neutral protease activity required for efficient islet release from the rat pancreas

Previous investigations clearly showed that the successful release of islets from the pancreas is mediated by both neutral protease (NP) and collagenase, consisting of subclasses I and II showing different capacities to cleave islets from the pancreas. Since no informations about the optimal ratio between class II and class I collagenase (II/I-ratio) are available yet, the present study sought to evaluate the efficient range for the II/I-ratio. METHODS: Following intraductal pancreas collagenase distension, rat islets were isolated utilizing 20 PZ-U Serva collagenase NB 1 and 1.0 or 0.4 DMC-U NP. After purification we determined the islet yield (IEQ), viability (trypan-blue exclusion) and function in diabetic nude mice. RESULTS: At 1.0 DMC-U NP, a II/I-ratio of 2.6, 1.5 or 0.7 yielded 2200 +/- 280, 2185 +/- 420, and 2205 +/-90 IEQ, respectively (ns). Viability varied between 70% and 80% (ns). Digestion time was significantly lowest (P < .05) using a II/I-ratio of 0.7. Utilization of 0.4 DMC-U NP resulted in a viability of >98% among all experimental groups (P < .001 vs 1.0 DMC-U). Islet yield decreased at a II/I-ratio of 2.6 (1520 +/- 120 IEQ, P < .05) and 1.5 (1780 +/- 130 IEQ, ns), but not at 0.7 (2310 +/- 160 IEQ, ns). Again, digestion time was lowest (P < .001) using a II/I- ratio of 0.7. Transplantation into diabetic nude mice demonstrated islet function in all experimental groups. CONCLUSIONS: NP significantly affects islet viability. This study indicates that the minimal amount of NP required for efficient islet cleavage depends on the II/I-ratio.     

Influence of donor age on bovine pancreatic islet isolation

BACKGROUND: Pancreatic islets from pigs are largely used for experimental studies. However, pancreas harvesting requires modification of conventional slaughtering to reduce ischemia time. It has been shown that bovine pancreatic islets can be more easily obtained and they show satisfactory in vitro and in vivo function. To improve the isolation procedure we compared the effect of bovine donor age on islet isolation. METHODS: Islets were isolated by collagenase digestion and sequential sieving from calves (6 months of age) and from adult bovine (> 16 months of age). After isolation the number of islet equivalents was calculated and histological and immunohistochemical studies performed. The purity and viability of islet for each preparation was also estimated. In vitro function of islets was evaluated by static insulin secretion assay, and alginate encapsulated islets were transplanted in streptozotocin-induced diabetic rats for in vivo functional evaluation. RESULTS: A significantly higher number of islets were obtained from calf pancreas, compared with adult bovine pancreas. Hystological examination showed intact morphologic features of islets. The purity of islet preparations was higher from calf pancreas than from adult pancreas. Cell viability, and insulin production in presence of high glucose concentration, were not affected by donor age. All animals receiving microencapsulated islets from calves showed normoglycemia for prolonged periods (17-40 days). CONCLUSIONS: These results indicate that pancreatic islet isolation is more efficient from juvenile bovine than from adult. Calf pancreas is a good and convenient source of tissue for massive islet isolation for experimental studies.     

Significant progress in porcine islet mass isolation utilizing liberase HI for enzymatic low-temperature pancreas digestion.

BACKGROUND: Frequent success in human islet isolation is prevented by the large variability of scarce organ donors; this favors the future utilization of pigs as donors for clinical islet xenotransplantation. Porcine-specific difficulties of islet isolation are attributed to the intrinsic fragility of islets during pancreas digestion. METHODS: To preserve islet integrity during efficient pancreas dissociation, porcine pancreata (n=48) were distended after cold storage with cold University of Wisconsin solution containing Liberase HI and digested at 24-28 degrees C using digestion-filtration. Pancreata distended with University of Wisconsin solution containing well-proven crude collagenase and digested at 32-34 degrees C served as controls (n=46). Monolayer Ficolldiatrizoate gradient purification was performed in a Cobe 2991. RESULTS: Purified yield of islet equivalents per pancreas (mean+/-SEM) was almost doubled by Liberase HI compared with crude collagenase (526,480+/-46,560 vs. 270,270+/-19,420; P < 0.0001) and also significantly increased comparing islet equivalents per gram of pancreas (4,210+/-320 vs. 2,640+/-245; P=0.0004). Islet integrity was better preserved during Liberase HI digestion compared with crude collagenase digestion as indicated by isolation index (2.1+/-0.1 vs. 1.4+/-0.1; P<0.0001). Purity, viability, and in vitro function of islets did not differ between experimental groups. Preserved in vivo function of islets isolated by Liberase HI was demonstrated after subcapsular transplantation into 16 diabetic nude rats. CONCLUSIONS: If the problems related to xenograft rejection and xenosis could be solved, low-temperature digestion of porcine pancreata using Liberase HI could serve as an essential prerequisite for successful 1:1 xenotransplantation of pig islets into type 1 diabetic human recipients.     

Oxygen tension and blood flow in relation to revascularization in transplanted adult and fetal rat pancreatic islets

We have previously recorded a decreased oxygen tension and blood flow in syngeneically transplanted rat pancreatic islets. The present study related measurements of oxygen tension and blood flow to the vascular density in such grafts implanted beneath the renal capsule. We also evaluated whether transplanted fetal islets are better revascularized than adult islets, and if the degree of revascularization is directly related to the islet vascular endothelial growth factor (VEGF) production. Tissue pO2 was measured using Clark microelectrodes, whereas islet graft blood flow was measured with laser-Doppler flowmetry. The vascular density of endogenous and transplanted islets was quantified in histological specimens stained with the lectin Bandeiraea simplicifolia (BS-1). Tissue pO2 in the transplanted adult and fetal islet grafts was similar and markedly lower than in the endogenous islets. The blood perfusion of both the adult and fetal islet grafts was 60-65% of that in the renal cortex. Administration of D-glucose did not affect tissue pO2 in either the endogenous or transplanted islets, nor graft blood perfusion. The number of capillaries found in the transplanted adult and fetal islets was similar and markedly lower than in endogenous islets. However, in the connective tissue stroma, which constituted approximately 20% of all islet grafts, the vascular density was higher than in the corresponding endocrine parts of these grafts. Incubated adult islets released higher amounts of VEGF than fetal islets. In conclusion, the previously described low oxygen tension of syngeneically transplanted adult rat islets is related to a low vascular density. Similar low oxygen tension and vascular density are seen in grafted fetal islets. The amount of VEGF production does not correlate to the degree of revascularization of the grafts.     

Studies of the isolation, viability, and preservation of purified islets after surgical pancreatectomy in large pigs

Abstract: Previous studies have shown separation of mass quantities of islets from slaughterhouse pig pancreases. In these studies, we examined the isolation, viability, and preservation of pig islets obtained by surgical pancreatectomy. Pigs aged 6 to 30 months weighing 100 to 200 kg were subjected to laparotomy under general anesthesia. The splenic lobe of pancreas was mobilized without warm ischemia. Islets were immediately isolated by collagenase perfusion through the duct, automated dissociation, and Ficoll purification. Yields of islets (150 μm size) were 8500 to 9300/g for two different collagenases before purification. Purification yielded 90% pure islets, but yields decreased to 400 to 600/g. Perifusate insulin release was biphasic after glucose/theophylline with 3 to 5-fold stimulation. Following culture/cryopreservation marked islet losses occurred but viability was preserved. Quantities of 1,500 islet equivalents resulted in euglycemia in nude mice. These data show that mass quantities of viable islets can be isolated after pancreatectomy, but there is marked islet loss during purification and subsequent preservation due to inherent islet fragility.     

Influence of collagenase loading on long-term preservation of pig pancreas by the two-layer method for subsequent islet isolation

BACKGROUND: The introduction of the two-layer method (TLM) for long-term human pancreas preservation revealed the enormous potential of TLM to improve graft function of isolated islets. It is still unclear whether pig islets can be successfully isolated from pancreases after prolonged cold ischemia. To clarify this question, pig pancreases were subjected to 7-hour preservation by University of Wisconsin solution (UWS) storage or TLM. Another aim was to verify whether TLM can be synergistically combined with intraductal collagenase injection before cold storage. METHODS: After intraductal flush with UWS, organs were distended with 4.4 PZ-U/g of UWS-dissolved collagenase NB-8 and neutral protease adjusted to respectively 1.1, 0.2, 0.5, or 0.8 DMC-U/g for pancreases freshly procured (n=6) or distended with enzymes before (TLM preloaded, n=7) or after cold storage (UWS storage, n=4; TLM postloaded, n=10). RESULTS: Purified islet yield decreased from 429,200+/-86,700 islet equivalents (IEQ) in unstored pancreases to respectively 37,670+/-19620, 210,400+/-22900 and 238,000+/-26600 IEQ in UWS-stored (P<0.01), TLM-preloaded, or postloaded organs (P<0.05). Purity (>90%), viability (>95%), and insulin content were not different between groups. Islets from UWS-stored pancreases fragmented extensively, preventing further assessment of in vivo function. Compared with other experimental groups, islets from TLM-preloaded organs were characterized by enhanced basal and stimulated insulin release. Sustained normoglycemia was observed in diabetic nude mice transplanted with islets from TLM-postloaded or unstored pancreases in contrast with transient function in TLM-preloaded islets. CONCLUSIONS: This study demonstrates that significant amounts of intact pig islets can be isolated after prolonged pancreas preservation by TLM. Enzyme administration before TLM preservation decreases islet graft function.     

Neonatal pig pancreata as a source of insulin-producing tissue for xenotransplantation

Abstract: Islet transplantation has been proven as a viable option for treating type 1 diabetics; however, widespread clinical application of this technique will necessitate an alternative source of insulin-producing tissue. Porcine pancreata may be a potential source of islets, but adult porcine islets are difficult to isolate, are fragile, and have a tendency to fall apart during culture. In this study, we investigated the feasibility of preparing functionally viable islet cells in large quantities from the neonatal pig pancreas. Following collagenase digestion and 7 days of tissue culture, a mean of 48,000 neonatal islet cell (NIC) aggregates were recovered per pancreas. These NICs were responsive to glucose in vitro and were capable of correcting hyperglycemia in alloxan-induced diabetic nude mice. These results indicate that the neonatal porcine pancreas can be used to produce large numbers of functionally viable islet cells.     

Portal Venous Oxygen Persufflation of the Donation after Cardiac Death pancreas in a rat model is superior to static cold storage and hypothermic machine perfusion

Success of clinical pancreatic islet transplantation depends on the mass of viable islets transplanted and the proportion of transplanted islets that survive early ischaemia reperfusion injury. Novel pancreas preservation techniques to improve islet preservation and viability can increase the utilization of donation after cardiac death donor pancreases for islet transplantation. Rat pancreases were retrieved after 30 min of warm ischaemia and preserved by static cold storage, hypothermic machine perfusion or retrograde portal venous oxygen persufflation for 6 h. They underwent collagenase digestion and density gradient separation to isolate islets. The yield, viability, morphology were compared. In vitro function of isolated islets was compared using glucose stimulated insulin secretion test. Portal venous oxygen persufflation improved the islet yield, viability and morphology as compared to static cold storage. The percentage of pancreases with good in vitro function (stimulation index > 1.0) was also higher after oxygen persufflation as compared to static cold storage. Retrograde portal venous oxygen persufflation of donation after cardiac death donor rat pancreases has the potential to improve islet yield.     

Effect of glucose toxicity on intraportal tilapia islet xenotransplantation in nude mice

BACKGROUND: Discordant xenogeneic islets transplanted intraportally into athymic nude rats experience primary non-function and are rapidly destroyed. Recently, it has been reported that adult porcine islets transplanted intraportally into nude mice are also rapidly destroyed and that this constitutes a new model for instant blood-mediated inflammatory reaction (IBMIR). METHODS: Tilapia (fish) islets were harvested, mechanically broken into mammalian islet-sized fragments, cultured for 48 h, and transplanted via the portal vein into athymic or euthymic mice. RESULTS: There were several groups of recipient mice. Streptozotocin-diabetic nude mice received 400 islets via the portal vein (n = 12). Recipients were killed when hyperglycemic (>200 mg/dl); livers and native pancreases were examined histologically. Mean graft survival time, based on function, was 5.4 +/- 1.2 days; at autopsy, histology showed occasional viable islets. We also performed a group of transplants in non-diabetic nude mice (n = 6) and then killed the recipients 2 or 4 weeks later; all had abundant viable, well-granulated islet grafts based on histology. Therefore, the intraportal environs in nude mice are not incompatible with discordant fish islets; rather, it appears as if hyperglycemia adversely affects the intraportal islet grafts (i.e. 'glucose toxicity'). To test this hypothesis, transplants were performed into non-diabetic nude mice and allowed to engraft for either 3 days (n = 6) or 10 days (n = 8) prior to injection of streptozotocin (200 to 220 mg/kg i.v.) to destroy the beta-cells in the recipients' native islets (n.b. tilapia islets are exceedingly resistant to streptozotocin); these recipients were followed for 28 days post-transplantation (or until hyperglycemic) and then killed for histology. Mean graft function exceeded 25 days for both groups and viable well-granulated, tilapia islets grafts were readily identified in all recipients; in all but one, the native pancreases were markedly beta-cell depleted -- confirming that normoglycemia was due to functional fish islet xenografts. CONCLUSIONS: Our results suggest that 'glucose toxicity' plays a role in the immediate demise of intraportal tilapia islet xenografts.     

Multiple donor allotransplantation. A new approach to pancreatic islet transplantation

Currently it is not feasible to isolate sufficient numbers of islets from a single pancreas for clinical transplantation. We examined whether small numbers of islets obtained from multiple donors could be used for transplantation. Islets were isolated from four inbred strains of mice (DBA/2, DBA/1, C3H, and A.SW) by a stationary collagenase digestion and Ficoll separation and transplanted into the renal subcapsular space of streptozotocin-induced diabetic B6AF1 mice. At least 200 handpicked islets were required to produce normoglycemia in syngeneic and allogeneic diabetic recipient mice. None of the mice given 50 islets became normoglycemic within 2 weeks postgrafting. When various numbers of purified islets from a single donor were transplanted, the survival was significantly better for 200-islet allografts than for 800-islet and 400-islet allografts. When a 200-islet composite graft was prepared from four donors (50 fresh handpicked islets from each donor), the survival of the composite graft as measured by sustained normoglycemia in nonimmunosuppressed recipients was dramatic, with 17 of 18 recipients maintaining normoglycemia indefinitely (greater than 200 days). Similarly, when islets isolated from four donors and cultured for various periods were mixed and transplanted (200 islets/recipient) all recipient mice (n = 8) enjoyed indefinite graft survival. Use of higher numbers of purified islets or crude islets in a composite multiple-donor islet allograft was less effective in achieving indefinite graft survival. Thus, transplantation of a composite graft made up with subtherapeutic numbers of islets from multiple histoincompatible donors to provide adequate therapeutic numbers is a practical solution to the lack of islet availability. In addition, composite islet grafts appear to possess immunological advantages, with significantly prolonged survival over that produced by single-donor islet grafts.     

Effective islet isolation method with extremely high islet yields from adult pigs

Achieving good islet isolation is one of the most important factors for successful islet transplantation. Porcine pancreas is suitable for islet isolation research due to its anatomical and physiological similarities to human pancreas. In this study, we evaluated a new porcine islet isolation method designed to maximize islet yield and compared it with our previous open pan method and the standard method using a Ricordi chamber (Ricordi method). We performed 15 porcine islet isolations, five each with the new method, the open pan method, and the Ricordi method. The new method features several important improvements. Pancreata remain uncut and are kept intact during collagenase intraductal injection, a large filtration chamber to handle whole pancreata, low concentration of collagenase (Liberase HI) for digestion, and large plastic containers for large-scale islet purification. All isolated islets were assessed for yield, purity, viability and in vitro function. Islets isolated with this new method were transplanted under the kidney capsules of SCID mice with chemically induced diabetes for in vivo functional assessment (n = 8). With the new method, we obtained on average more than 1,000,000 islet equivalents (IE) (1,236,266 +/- 213,486 IE) (mean +/- SE) before purification and 800,000 IE (879,815 +/- 222,729 IE) after purification from one adult pig. Islet yield per pancreas was significantly higher compared with our previous open pan method (30,666 +/- 11,532 IE, p < 0.01) and the Ricordi method (317,073 +/- 86,093 IE, p < 0.05). All mice, transplanted with 1000 islets from the new method, returned to normoglycemia within 4 days after transplantation. Our new method makes it possible to obtain extremely high porcine islet yield with good function. It should produce useful information for human islet isolation and transplantation, and might be applied to single donor clinical xenogeneic transplantation.     

Survival of an islet allograft deficient in iNOS after implantation into diabetic NOD mice

Proinflammatory cytokines play a major role in rejection of pancreatic islet allografts and in type 1 diabetes (T1D). In rodent islets, exposure to IL-1beta alone or combined with IFN-gamma induces expression of inducible nitric oxide synthase (iNOS). Inhibition of iNOS or a deletion of the iNOS gene has been shown to be protective in animal models of T1D. In the present study we tested the hypothesis that transplantation of pancreatic islets deficient in iNOS (iNOS-/-) would permit increased graft survival. Pancreatic islets isolated from wild-type (wt) mice and iNOS-/- mice were allogeneically transplanted beneath the kidney capsule of spontaneously diabetic NOD mice. When blood glucose increased above 12.0 mM after preceding normalization of hyperglycemia, animals were sacrificed. Histological examinations of grafts were performed and graft gene expression was analyzed by real-time PCR. Transplantations of the two types of islets could reverse hyperglycemia and the grafts functioned for on average 1 week posttransplantation. Morphological examination of both types of islet grafts showed immune cell infiltration around and within the grafts. Remaining endocrine cells could be observed in wt and iNOS-/- islet grafts. In the removed grafts iNOS-/- islet tissue contained higher mRNA levels of insulin, proinsulin convertases (PC-1 and PC-2), and IL-1beta compared to transplanted wt islets. The assessments of insulin, PC-1 and PC-2 mRNAs of the grafts suggest that the iNOS-/- islets may be more resistant to destruction in the transplantation model used; however, this was not sufficient to prolong the period of normoglycemia posttransplantation.     

Adaption of neutral protease activity for islet isolation from the long-term two-layer method-stored pig pancreas

BACKGROUND: Islet release from the pancreas is mediated by both collagenase and neutral protease (NP), a critical effector of islet integrity. To prove the hypothesis that adjustment of NP reduces islet damage after prolonged ischemia, adult pig pancreata were digested after 7-hour preservation by the two-layer method (TLM) using a 2-component enzyme blend consisting of collagenase NB-8 and NP. METHODS: After intraductal University of Wisconsin (UW) flush resected pancreata were distended with 4.4 PZ-U/g of UW-dissolved Serva collagenase either before (TLM-preloaded, n = 7) or after (TLM-postloaded, n = 10) cold storage, or for immediate processing (n = 6). NP was adjusted after preliminary experiments to respectively 1.1, 0.2, or 0.8 DMC-U/g for unstored, TLM-preloaded, or postloaded organs. RESULTS: Purified islet yield decreased from 3670 +/- 730 islet equivalents (IEQ)/g in unstored pancreata to 1800 +/- 180 and 2080 +/- 290 IEQ/g in TLM-preloaded or postloaded organs, respectively (P < .05). Although purity was always >90%, IEQ recovery was significantly decreased in TLM-preloaded pancreata. Quality control revealed consistently high viability as determined using trypan-blue exclusion (>95%) or formazan production. Compared with unstored organs (2.47 +/- 0.36; P < .05), glucose stimulation index was reduced in TLM-preloaded (1.48 +/- 0.15) and TLM-postloaded pancreata (1.81 +/- 0.20). Normoglycemia in diabetic nude mice transplanted with islets from TLM-preloaded pancreata was transient in contrast to sustained function in the other groups. CONCLUSIONS: Significant amounts of viable pig islets can be isolated after prolonged TLM preservation by reducing NP activity. Nevertheless, early enzyme administration prior to long-term storage deteriorates islet graft function.     

The ratio between collagenase class I and class II influences the efficient islet release from the rat pancreas

BACKGROUND: Previous studies indicated different roles of collagenase class I, class II and neutral protease in the enzymatic islet release from pancreatic tissue. Because no information has been available, this study was aimed to investigate the isolation efficiency of different ratios between collagenase class II and I (C-ratio) in the rat pancreas serving as model for the human pancreas without being restricted by the large variability observed in human donors. METHODS: Rat pancreata were digested using a marginal neutral protease activity and 20 PZ-U of purified collagenase classes recombined to create a C-ratio of 0.5, 1.0, or 1.5. Collagenase efficiency was evaluated in terms of isolation outcome and posttransplantation function in diabetic nude mice. RESULTS: The highest yield of freshly isolated islets was obtained using a C-ratio of 1.0. Purity and fragmentation of freshly isolated islets were not influenced by the C-ratio. After 24-hr culture performed for quality assessment, a marginal but significant reduction of viability was observed in islets isolated by means of a C-ratio of 0.5 and 1.5. Islet in vitro and posttransplantation function revealed no negative effect mediated by different C-ratios. CONCLUSIONS: The present study demonstrates that the C-ratio is of significant relevance for the outcome after enzymatic rat islet isolation. The data indicate further that purified collagenase class I or class II does not damage islet tissue even if used in excess. The present study can serve as a start for subsequent experiments in the human pancreas.     

大量人胰岛分离技术的改进及相应胰岛功能的检测

目的 通过对人胰岛分离技术的改进以获得大量高活力胰岛并检测其功能。方法采用经典及改良技术分离、纯化28例人胰岛。胰岛收获量以胰岛当量(IEQ)表示。胰岛功能测定分别为体外测定胰岛素/DNA比率、静止葡萄糖刺激试验(SGS)及将胰岛移植至糖尿病裸小鼠的体内功能鉴定并行糖耐量试验,连续测血糖水平及其体内C肽浓度。结果 前13例采用经典方法,平均每个胰腺收获49123 IEQs,平均846 IEQs/g胰腺组织,平均纯度为87％。改进技术后15例的分离结果则分别为:501813 IEQs/胰腺,7003 IEQ/g胰腺组织及纯度89％。体外胰岛素刺激试验结果表明分离纯化后的人胰岛有正常功能,将12次分离得到的胰岛分别移植至34只糖尿病裸鼠肾包膜下,其中29只糖尿病裸鼠于12h内血糖恢复正常且糖耐量试验接近正常鼠,血中C肽水平亦接近正常。结论 采用改进的人胰岛分离方法,可以获得大量高活力的具有正常功能的胰岛,为同种异体胰岛移植用于临床奠定了必要的基础。     

A model to evaluate toxic factors influencing islets during collagenase digestion: the role of serine protease inhibitor in the protection of islets

The recovery of all of the islets contained in a pancreas is the goal of islet isolation for transplantation. This study reveals an environment that injures the isolated islets during digestion and proposes a new model for optimal islet isolation. Islets were isolated from Wistar rat pancreases by stationary collagenase digestion while the digestion time was varied at 15, 30, 60, and 120 min. The digested pancreas and islets were analyzed histologically and adenosine nucleotides were measured. Overnight cultured islets (40 islets) were cocultured for 30 min with the supernatants obtained from pancreatic collagenase digestion at different digestion periods in order to assess the toxic environment. The peak yields of islets were obtained at 30 min of digestion. The histological study of digested pancreas showed that the exocrine cells lost their cellular integrity at 120 min of digestion, but the islet cells were left intact. Accordingly, the ATP levels of the pancreatic tissue decreased during the digestion period. The coculture experiment demonstrated that the islets cultured with the supernatants from the collagenase digestion showed digestion time-dependent disruption of the cellular integrity of islets in accordance with a rapid decrease of ATP levels in the islets. The addition of serine protease inhibitors into this coculture clearly showed protection of islets, which maintained high ATP levels in association with intact membrane integrity as assessed by AO/PI staining. Morphological deterioration of islets as well as a marked ATP decrease was evident in the entire digested pancreas as well as in islets cocultured in the supernatants from the collagenase digestion. Various factors toxic to the islets can therefore be analyzed in future experiments using this coculture model for obtaining a good yield of viable islets.     

Infant human pancreas. A potential source of islet tissue for transplantation.

Twelve pancreases from human infants one year old or less were analyzed for tissue insulin and amylase content before and after dispersal of pancreatic fragments by mincing and collagenase digestion. Tissue insulin and amylase content provide an index of pancreatic islet mass and exocrine digestive enzyme content, respectively. The results were compared with similar anaylses performed on juvenile and adult human pancreases before and after islet isolation and on intact and dispersed neonatal rat and adult rat pancreas. Infant human pancreas has an average tissue insulin concentration of 1,128 mug./gm. of tissue and a total insulin content of 1,718 mug/pancreas, as against values of 140 mug./gm. of tissue and 7,209 mug./pancreas for adult human pancreas. Average tissue amylase concentration is 0.24 mg./gm. of tissue in infant human pancreas and 3.0 mg./gm. of tissue in adult human pancreas. The insulin/amylase ratio in infant pancreas is 4,800, as against 46 in the adult pancreas. Neonatal rat pancreas, which can be dissociated and transplanted without separation of islet and exocrine components, has a similarly high tissue insulin and low tissue amylase content when compared with adult rat pancreases. Infant human pancreas has a total islet mass 24 per cent that of an adult human pancreas, and neonatal rat pancreas has a total islet mass 11 per cent of that of an adult rat pancreas. One neonatal rat pancreas prepared by minimal collagenase digestion can cure diabetes when transplanted via the portal vein to a rat. Following dispersal of infant human pancreas by collagenase digestion, the islet content and the insulin/amylase ratio of the recovered tissue equals or exceeds that which usually can be isolated from adult cadaver pancreases. Infant human pancreas is a rich source of islet tissue that is relatively uncontaminated by exocrine digestive enzymes. After dispersal, infant human pancreas may be ideal for transplantation to selected diabetic patients.     

Successful reversal of diabetes in nude rats by transplantation of isolated adult human islets of Langerhans

A method is described in which the viability of isolated adult human islets of Langerhans can be assessed in vivo. The Rowett nude rat, made diabetic with streptozocin (STZ), has been used as the islet recipient in these studies. Although these animals are athymic and are able to accept xenogeneic grafts for prolonged periods, they are very susceptible to dehydration and infection once made diabetic. Therefore, a considerably shortened diabetes induction period was used. The basis of the study was to prepare pure adult human pancreatic islets that were cultured for 48 h. Nude rats were given 80 mg/kg i.v. STZ during islet isolation and were transplanted with 800-1000 islets under the renal capsule at 48 h. To monitor islet function, animals were bled regularly for random blood glucose measurements and were given a glucose tolerance test at day 20. The kidney containing the graft was removed on day 21 to allow histological assessment of the graft and to confirm that glucose control was due to the transplanted islets and was not secondary to reversion of the animal's own islets. Seven rats were transplanted, and five were deemed to have received viable human islets. Two rats that received islets from the same donor did not reverse their diabetes and were found by histology to have vacuolated islet structures with scant insulin-staining tissue under the kidney capsule. This method allows a definitive judgment of the ability of isolated adult human islets to reverse diabetes.