玻璃化法冷冻保存对人胚胰岛的影响

目的:观察玻璃化法对人胚胰岛冻存复苏后形态功能的影响.方法:取胎龄18-28wk中期水囊引产死胎,用胶原酶分离胰岛后,分别采用传统微机程控缓慢降温和玻璃化超快速降温方法冷冻保存胰岛,复苏后,从胰岛形态、超微结构、胰岛素分泌功能方面比较两种冻存方法的效果.结果:两种方法冷冻保存的胰岛,复苏后形态保持完整,电镜下胰岛B细胞分泌颗粒和线粒体丰富,线粒体嵴规整,胰岛素释放试验中,二者的胰岛素水平与冻存前无明显差异(P>0.05),胰岛素基因表达水平与冻存前相似.结论:玻璃化法冷冻保存胰岛能够维持细胞的正常结构和功能,其冷冻效果与微机程控缓慢降温相比无明显差别.     

传代和冻存对胎肺间充质干细胞生长及分化的影响

目的 观察传代和冻存对胎肺间充质干细胞（MSCs）生长及分化的影响.方法 以胶原酶消化法自流产胎儿肺组织中分离MSCs,在体外进行传代扩增、冻存、复苏;然后,以相差显微镜观察细胞形态学变化,以流式细胞仪检测表型变化,以神经分化诱导方案研究其向神经元样细胞分化的潜能.结果 经传代和冻存后,人胎肺MSCs的增殖能力、形态和表面标志物的表达均无明显变化,高表达MSCs特异性标志物,不表达造血和内皮细胞标志物,在适宜的神经诱导方案作用下可高效分化为神经元样细胞.结论 传代和冻存对人胎肺MSCs的形态、表型和向神经样细胞的分化潜能无明显影响,胎肺MSCs可作为细胞移植治疗神经系统疾病的备选来源.     

人胚胎关节软骨细胞库的建立

目的：体外建立人胚胎关节软骨细胞冻存、复苏的稳定技术，为软骨组织工程提供大量的、具有软骨细胞生物活性的种子细胞。方法：将传代培养的人胚胎关节软骨细胞用含10%DMSO、50%胎牛血清的IMDM培养液悬浮后，置于-196℃的液氮中长期保存，建立人胚胎关节软骨细胞库。然后将细胞库中的冻存细胞进行复苏培养，观察其生长状况，并测定软骨细胞中DNA及糖醛酸的含量。结果：冻存软骨细胞复苏后仍保持旺盛的增殖与分泌细胞外基质的功能。结论：关节软骨细胞库的建立，可以长期保存培养的软骨细胞。     

人芽囊原虫保存方法的初步研究

目的 观察冻存剂、温度等因素对人芽囊原虫活力的影响，探索理想的人芽囊原虫保存方法。 方法 从患者阳性粪便中分离人芽囊原虫，分装到2 ml无菌冻存管内，分别加入10%二甲基亚砜（dimethylsulfoxide, DMSO）、40%丙三醇（GL）和15%乙二醇（EG）作冻存剂，放置于不同的温度下保存，用台盼蓝染色法和原虫培养法测定细胞的活力和增殖能力。 结果 虫体在室温（18 ℃～20 ℃）下可存活3周，在4 ℃～6 ℃存活不到1周。加入冻存剂后，置－20 ℃和液氮（－196 ℃）下可存活3个月以上。用40% GL作冻存剂，于液氮低温下冻存的虫体保存半年后复苏， 活力仍达41.7%，培养72 h后多见分裂相细胞。 结论 应用40% GL作为冻存剂， 在液氮中低温保存人芽囊原虫效果较佳。 4 ℃不适于保存人芽囊原虫。     

猪胰岛异种移植治疗Ⅰ型糖尿病

猪胰岛异种移植治疗Ⅰ型糖尿病陈冰，祝希媛近年来，移植的观念已被人们所接受。针对Ⅰ型糖尿病病人，胰岛移植将是比较彻底的治疗方法。我国是世界上进行人胎胰岛移植数量最多的国家，但由于人胎胰缺乏，人们已考虑猪做为供体的可能性。猪的胚胳发育过程与人相似，胎猪４...     

超速玻璃化法冻存小鼠肝细胞球形聚集体的初步研究

现有的肝细胞冻存法是将分散的肝细胞用于冻存.从肝组织消化分离的肝细胞,其细胞完整性和功能都会受影响.有报道用肝细胞球形聚集体培养,效果优于用分散肝细胞培养[1].玻璃化法冻存细胞组织,由于不形成冰晶,对细胞损伤较小,已成功用于冻存胚胎、卵巢、胎肝组织和脑细胞[2-5].本研究将小鼠肝细胞预培养成球形聚集肝细胞,用改进的超速玻璃化法冻存,解冻后与含胶原培养液混合、培养,通过检测培养上清液中ALT、AST、乳酸脱氢酶(LDH)浓度判断肝细胞受损漏出情况,检测白蛋白( Alb)浓度判断肝细胞合成功能.对照组用单层贴壁培养分散肝细胞做同样冻存和检测.     

成人脂肪肝整肝肝细胞分离及大量冻存的实验研究

目的建立成人脂肪肝整肝肝细胞的分离以及人肝细胞大量冻存技术,为生物人工肝提供稳定的人肝细胞来源。方法采用胶原酶经肝静脉逆行灌注的方法分离成人重度脂肪肝的整肝肝细胞,并比较常规冻存和程序冻存后肝细胞在细胞活性、贴壁率、LDH漏出量及白蛋白合成能力的差异。结果采用添加N-乙酰半胱氨酸(NAC)的胶原酶灌注液分离肝细胞的产量为(7.4±0.5)×10~6 cells/g肝组织,活性为(81.4±3.4)%,而未添加NAC组的肝细胞产量为(5.6±0.8)×10~6 cells/g肝组织和活性为(67.3±5.0)%,差异均有统计学意义(P0.05)。分离的人肝细胞采用程序冻存后在细胞活性、贴壁率及白蛋白合成能力方面均相应高于常规冻存组(P0.05),LDH漏出量低于常规冻存组(P0.05)。结论应用添加NAC的胶原酶灌注液经肝静脉逆行灌注可提高脂肪肝整肝肝细胞分离的活性及产量,采用程序冻存的方法可以提高冻存人肝细胞的活性,满足生物人工肝对肝细胞的需要。     

大鼠自体皮下移植卵巢的冻存剂选择

目的观察不同冻存剂对大鼠卵巢组织的冻存效果,以选择良好的卵巢组织冻存剂。方法 48只成年雌性Wistar大鼠随机分为实验组和对照组。实验组大鼠切除卵巢后于不同的冻存液中冻存,根据选用冻存剂不同将实验组分为A、B、C、D组,A组冻存剂为二甲基亚砜（DMSO）＋丙二醇（PROH）＋胎牛血清（FBS）＋蔗糖（S）＋乙酰胺（AC）、B组为DMSO＋PROH＋FBS＋S、C组为DMSO＋PROH＋FBS、D组为PROH＋S＋AC。各组卵巢在液氮中冻存2周后解冻并行自体皮下移植,3个月后自腹主动脉取血检测血清雌二醇（E2）并进行移植卵巢组织切片观察。对照组为E和F组,E组为正常对照组,F组为新鲜卵巢移植组。结果 A、B、C、D组与E组相比,各级卵泡均有不同程度的损伤和数量减少;A组卵巢组织中具有完整结构的初级卵泡和次级卵泡数量多于B、C、D组。A、B、C、D组血清E2水平低于E组（P均〈0.01）,B、C、D三组血清E2水平低于F组（P均〈0.01）,A组血清E2水平明显高于B、C、D三组（P均〈0.05）。结论冻存剂DMSO＋PROH＋FBS＋S＋AC对大鼠卵巢的冷冻效果较好,移植后的卵巢组织有较好的功能。     

用于生物人工肝的大量猪肝细胞低温冻存技术

目的：比较大量猪肝细胞程序冻存与常规冻存的效果。方法采用二步法胶原酶经肝静脉逆行灌注的方法分离猪肝细胞,分离的肝细胞根据冻存方法与冻存袋的不同分5组：a 组,50 mL 冻存袋,程序冻存;b 组,100 mL 冻存袋,程序冻存;c 组,50 mL 冻存袋,常规冻存;d 组,100 mL 冻存袋,常规冻存;e 组,新鲜分离的肝细胞。各冻存组解冻后,比较各组的肝细胞活性、贴壁率、LDH 漏出量及白蛋白合成能力。结果新鲜分离的肝细胞产量为（1．8＆#177;0．4）1010/肝,活性高达（90．5＆#177;1．7）％,培养后贴壁率为（70．5＆#177;8．5）％。采用程序冻存的 a、b 组在肝细胞活性、贴壁率、LDH 漏出量及白蛋白合成能力方面明显优于常规冻存的 c、d 组（P＜0．05）,但与 e 组新鲜分离的肝细胞相比,其解冻后肝细胞的活性、贴壁率及蛋白合成能力均降低（P＜0．05）,LDH 漏出量增加（P＜0．05）。相同冻存方法条件下,50 mL 袋装与100 mL 袋装冻存效果差异无统计学意义（P＞0．05）。结论采用袋装程序冻存法大量冻存猪肝细胞可满足生物人工肝对肝细胞活性及数量的要求。     

卡氏肺孢子虫冻存复苏后增殖的实验研究

目的　研究卡氏肺孢子虫 (Pc)冻存复苏后在细胞培养的增殖情况。观察冻存Pc复苏后接种大鼠Pc感染发生率。方法　从免疫抑制大鼠肺中分离Pc,以不同浓度二甲亚砜和甘油作冻存液冻存Pc 3个月和 1年。解冻后 ,将其分别接种于HepG - 2细胞系和已免疫抑制 2周的大鼠。观察和比较冻存 3个月和 1年的Pc增殖情况及大鼠肺部Pc感染情况。 结果　以 15 %二甲亚砜冻存的Pc增殖幅度最大 ,第 4、5d可增长 7倍左右。冻存 1年与冻存 3个月的Pc增殖幅度无明显差异。接种冻存Pc后的大鼠 ,肺部Pc感染的发生率是对照组 4 .5倍。结论　 15 %二甲亚砜冻存的Pc复苏后增殖率最高。冻存 1年与冻存 3个月的Pc复苏情况显著性差异。用冻存Pc接种免疫抑制大鼠 ,可使诱发大鼠Pc感染模型时间明显缩短。     

胎鼠胰腺干细胞体外对胰岛的保护作用观察

目的观察胎鼠胰腺干细胞体外对胰岛功能的保护作用。方法将孕16d的sD大鼠胎鼠胰腺干细胞分离、纯化、培养传代,免疫细胞化学法及流式细胞术鉴定;分离纯化sD大鼠胰岛,双硫腙鉴定后分为A组和B组,分别行单纯胰岛培养及胰岛与胰腺干细胞联合培养14d,期间观察胰岛形态变化、检测胰岛存活率及细胞凋亡率,ELISA法检测胰岛素分泌量、计算刺激指数。取两组培养7d后悬浮生长的胰岛移植入糖尿病大鼠左肾包膜下,术后每天尾静脉采血以快速血糖测试仪检测血糖。结果胎鼠胰腺干细胞培养传代3代后免疫细胞化学可见巢蛋白（Nestin）阳性细胞,流式细胞术测定其含量占74．1％;培养第7、14天时B组胰岛存活率显著高于A组（P均〈0．01）,培养第7天时B组胰岛细胞凋亡率显著低于A组（P〈0．05）;培养第7、14天时B组高糖刺激后胰岛素分泌量及刺激指数均显著高于A组（P均〈0．01）;B组移植后大鼠血糖第5天降至正常。结论胎鼠胰腺干细胞与胰岛联合培养可明显延长胰岛体外存活时间并使其保持良好的活性。     

Persistence of peptidylglycine alpha-amidating monooxygenase activity and elevated thyrotropin-releasing hormone concentrations in fetal rat islets in culture

Two experimental systems were used to investigate the relationship between TRH content and peptidylglycine alpha-amidating monooxygenase (PAMase) activity of the neonatal rat pancreatic islets: freshly isolated islets from rats aged 1-14 days, and fetal islets maintained in culture for 3 weeks. TRH was present in freshly isolated islets and in newly formed fetal islets in culture. However, while the TRH concentration in freshly isolated islets measured by RIA followed the same ontogenic pattern as that in the total pancreas, peaking during the first week of life (78 pg/micrograms DNA on day 4) and decreasing thereafter to reach 4 pg/micrograms DNA on day 14, the TRH content of fetal islets in culture did not decrease with time (65 pg/micrograms DNA on day 1 and 80 pg/micrograms DNA after 3 weeks in culture). Similarly, using immunocytochemistry, immunoreactive cells were only seen at day 2 in freshly isolated islets. In contrast, in fetal islets, TRH cells were present throughout the culture period. In both experimental systems, TRH was localized in beta-cells. PAMase activity paralleled TRH concentration, peaking during the first week of life in freshly isolated islets and remaining unchanged in the fetal islets in culture. PAMase activity is, therefore, present in the endocrine pancreas. The results suggest that PAMase activity is a rate-limiting step in TRH biosynthesis in this tissue.     

Rat fetal islets as a useful model for the study of insulin release failure

To study the mechanism of imparied insulin secretion from rat fetal islets, the insulin responsiveness of islets from fetuses (day 21.5 of gestation) to a variety of secretagogues was compared with that of adult rat islets. Forskolin (30 microM)-induced insulin release from fetal and adult islets was 2.7-and 2.5-fold higher, respectively, than that from islets treated with 5.6 mM glucose alone. The effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA) (200 nM) were also similar in fetal and adult islets. Thus, the responsiveness to forskolin and TPA showed no significant difference in adult and fetal islets. A synergistic effect of combinations of various insulin secretagogues was observed in adult islets; however, a weak synergistic effect was present with gliclazide plus TPA only in fetal islets. After islets were cultured in RPMI 1640 (containing 11.1 mM glucose), gliclazide-, forskolin-, and TPA-induced insulin release reached the levels obtained in adult islets. However, the synergistic effect of gliclazide and TPA disappeared after culture of the islets. These results suggest that the poor insulin secretion from fetal islets is not due to a defect in the activating system of either cAMP or C-kinase, but to the immaturity of the interaction of those messenger systems.     

High Recovery of Functional Islets Stored at Low and Ultralow Temperatures

BACKGROUND: Poor recovery of islets upon cryopreservation is the main hurdle in islet banking. Pancreatic islets have a poor antioxidative defense mechanism, and exposure of islets to low temperature leads to oxidative stress. AIM: We aimed to investigate whether known compounds such as metformin, γ aminobutyric acid (GABA), docosahexanoic acid (DHA), or eicosapentaenoic acid (EPA) alone or in combination are capable of reducing oxidative stress for better islet recovery upon storage at suboptimal temperatures. METHODS: Islets isolated from mouse pancreas were stored at low temperature (4°C) for 15 days and at ultralow temperature (-196°C) for 30 days with or without additives. After revival from cold storage, islets were assessed by using three methods: (1) specificity by dithizone (DTZ), (2) viability by fluorescein diacetate/propidium iodide (FDA/PI) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay, and (3) functionality by glucose-stimulated insulin secretion (GSIS). The oxidative status of the islets stored at suboptimal temperatures was determined by both intracellular free radical release (fluorometric analysis) and lipid peroxidation (enzymatic determination). RESULTS: Supplementation with additives led to an improvement in islet survival upon storage at suboptimal temperatures, without depletion of insulin secretory activity, which was comparable to that of controls. The additives acted as cryoprotectants and antioxidants as revealed by high recovery of viable islets and reduction in total reactive oxygen species (ROS) and malonidealdehyde (MDA), respectively. CONCLUSIONS: Our results demonstrate for the first time that supplementation with EPA, DHA, and metformin may lead to higher islet recovery from -196°C storage, enabling proper islet banking.     

Effect of retinoic acid on glucokinase activity and gene expression and on insulin secretion in primary cultures of pancreatic islets.

Retinoic acid has manifold effects on pancreatic beta-cells. Previously we reported that retinoic acid increases glucokinase activity and messenger RNA (mRNA) levels in the insulinoma cell line RIN-m5F; however, we could not rule out the possibility that the effect of retinoic acid on RIN-m5F glucokinase was inherent to the cell line or related to its differentiating capacity. In this report, we demonstrate that physiologic concentrations of retinoic acid stimulate glucokinase activity in both fetal islets and differentiated adult islets in culture. In the adult tissue, the response to the retinoid was less pronounced, achieving about half of the maximal effect produced on the fetal tissue. Using the branched DNA (bDNA) assay, a sensitive signal amplification technique, we detected relative increases in glucokinase mRNA levels of 51.8+/-13.3% and 62.8+/-16.1% at 12 and 24 h, respectively, in adult islets treated with] 10(-6) M retinoic acid. In fetal islets, increases of 55+/-14.9% and 107+/-30.5% at 12 and 24 h, respectively, were observed. In transfected fetal islets, retinoic acid increased the activity of the -1000 kb rat glucokinase promoter by 51.3%. Because glucokinase activity controls insulin secretion, we also investigated the effect of retinoic acid on insulin secretion. Treatment with 10(-6) M retinoic acid for 24 h increased insulin secretion in both fetal and adult islets; however, the increases on insulin secretion were more pronounced in the mature islets; in contrast, retinoic acid produced higher levels of insulin mRNA in the fetal islets. These data show that retinoic acid increases pancreatic glucokinase in cultured islets and that the mechanism may involve a stimulatory effect on the glucokinase promoter.     

Insulin secretagogues,but not glucose,stimulate an increase in [Ca2+]i in the fetal human and porcine beta-cell

Fetal pancreatic beta-cells release insulin poorly in response to glucose; however, the cellular mechanism for this is unknown. By using fura-2 to measure changes in the cytoplasmic free Ca(2+) concentration in beta-cells, we examined human/porcine fetal islet-like cell clusters (ICCs) and human adult islets for the presence of functional K(+)(ATP) and voltage-activated Ca(2+) ion channels. The effects of glucose, glyceraldehyde, leucine, KCl, and the channel effectors glipizide and BAY K8644 were studied. In fetal human/porcine ICCs and adult islets, KCl, glipizide, and BAY K8644 increased [Ca(2+)](i). Both glucose and glyceraldehyde increased [Ca(2+)](i) in islets but had no effect on ICCs. Leucine increased [Ca(2+)](i) in islets and porcine but not human ICCs. We hypothesize that the beneficial effect of leucine in fetal porcine, but not human ICCs, is attributable to time-dependent maturation of the beta-cells, because porcine ICCs examined were at 87% of the gestational period, and human ICCs were at 42%. Our data demonstrate that both K(+)(ATP) and voltage-activated Ca(2+) channels, required for glucose-stimulated increase in [Ca(2+)](i), are functional early in gestation. This suggests that the cause of the immaturity of fetal human/porcine beta-cells is at a more proximal step of glucose-induced metabolism than the channels on the cell surface.     

Insulin,glucagon and somatostatin secretion by cultured islets from normal and diabetic hamsters

Summary The interhormonal relationship within the pancreatic islets have been studied by previous investigators, but the cellular interplay and the sequence of events in the islet cell's response to stimulators has remained unclear. In the present study, pancreatic islets were isolated by collagenase digestion from normal and streptozotocin-diabetic hamsters the latter being maintained with insulin treatment. The diabetic animals were used to provide A- and B-cell enriched islets. The islets from normal and diabetic hamsters were cultured in medium 199 plus 10% fetal calf serum with 0.8 or 5 mg/ml glucose. The cultures were maintained for up to seven days     

Neonatal rat islets of Langerhans and fetal rat pancreas Isolation,immunohistochemical, functional,and autoradiographic evaluation

The aim of this study was to develop an optimal isolation technique for neonatal rat islets of Langerhans, to perform functional evaluation in vitro, to evaluate immunohistochemically isolated rat islets and fetal rat pancreata after a variable period of culture, and to study growth potentials by means of autoradiography. The islets were isolated using minor modifications of standard procedures including collagenase and DNase. Islets were separated on a discontinuous Percoll gradient. The maximum yield of islets amounted to 240 per pancreas. Fetal pancreata from rats were cultured under similar conditions as neonatal islets to compare their insulin secretory capacity after different periods of culture. The insulin secretion increased gradually, and isolated islets achieved a similar secretion potential to adult rat islets. The mitotic activity of both islets and fetal pancreata was confirmed using tritiated thymidine. The isolation procedure was found suitable for producing well-functioning islets, which could be kept in culture for a period of about 1 month without deterioration in their insulin secretory capacity. The gradual increase in insulin secretory capacity of islets and fetal pancreata was due, in part, to hyperplasia and not just hypertrophia. Autoradiographical evaluation revealed a high mitotic activity after culture, in particular of fetal pancreata. Fetal pancreata cultured for about 10 days showed a phenomenon of budding endocrine cells at the organ surface. A high mitotic activity was found in these buds.     

Peptidergic regulation of maturation of the stimulus-secretion coupling in fetal islet beta cells

The stimulus-secretion coupling of the insulin-producing pancreatic islet beta cell is subject to functional maturation during fetal life. We studied the maturation of a glucose-responsive insulin release from fetal rat islets and specifically investigated the impact of peptidergic regulation. To this end, islets were isolated from 21-day-old fetal rats and maintained for 7 days in tissue culture at 3.3 or 11.1 mM glucose and various supplements. In islets cultured in low glucose, acutely raising the ambient glucose concentration to 16.7 mM evoked a modest stimulation of short-term insulin release that was more pronounced in islets maintained in high glucose. Moreover, the insulin content was much higher in islets cultured in high than in low glucose. Culture with growth hormone (GH) markedly amplified both basal and stimulated short-term insulin secretion from islets maintained in either low or high glucose. Additionally, GH significantly elevated the insulin content in islets maintained in low glucose. Transforming growth factor alpha (TGF-alpha) increased basal, but not glucose-stimulated, insulin release and insulin content in islets cultured in low glucose. Gastrin, expressed in islets during fetal life, did not affect basal or glucose-stimulated insulin release, or insulin content, in islets maintained in either low or high glucose. The addition of gastrin to TGF-alpha did not affect the results obtained with the latter peptide. Gastrin-releasing peptide failed to influence basal or glucose-responsive insulin secretory rates, and insulin content, at either glucose concentration during culture. The somatostatin analog Sandostatin (octreotide acetate) neither influenced basal nor stimulated short-term insulin release at any glucose concentration present during culture, whereas the hormone significantly decreased the insulin content of islets cultured in high glucose. Pancreastatin, produced by porcine islet beta and delta cells, failed to influence basal or glucose-responsive insulin secretory rates, and islet insulin content, at either glucose concentration during culture. Culture with gastric inhibitory peptide (GIP) or glucagon-like peptide I (GLP-1), two proposed incretins, did not affect short-term insulin secretion in response to 3.3 or 16.7 mM glucose irrespective of the ambient glucose concentration during culture. To the contrary, GLP-1, but not GIP, increased the content of insulin in islets cultured in low glucose. We conclude that islet beta-cell differentiation and functional maturation of the stimulus-secretion coupling can be modulated in vitro in fetal rat pancreatic tissue by peptidergic regulation and glycemic stimulation. We suggest that GH and TGF-alpha stimulate, while somatostatin, through paracrine interaction, may inhibit, these processes. These effectors may be of regulatory significance in the in vivo development of glucose-sensitive beta cells, and defects in these mechanisms may result in glucose intolerance in adult subjects.