大肠癌中口服化疗的作用（文献综述）

大肠癌口服化疗药主要指氟嘧啶类前体药物，吸收后通过一次或多次代谢转变成5－氟尿嘧啶（5－Fu)，发挥抗癌作用。口服化疗在临床应用中疗效高、不良反应少、给药方便，宜于老年肿瘤患者和家庭化疗，成为大肠癌辅助治疗的一个新趋势。     

硝苯啶滴鼻用于颅内手术控制性降压的初步报告

硝苯啶滴鼻用于颅内手术控制性降压的初步报告胡毅平，刘林汉，浦惠珍，王雁娟我们试用鼻腔分次滴入硝苯啶（ＮＩＦ）混悬液，配合体位调节及加深全麻，必要时辅以少量ＳＮＰ的方法，用于需行控制性降压的脑肿瘤病人。现将初步体会报告于下。临床资料和方法本组８例均为颅...     

白细胞介素-2及其受体与胰岛移植排斥反应发生的相关性及其意义

目的：探讨检测血清中白细胞介素－2（IL－2）及其受体（SIL－2R）的含量对异种胰岛移植排斥反应中的意义。方法：昆明小鼠为供体，Wistar大鼠为受体。分4组：A组为糖尿病对照组，B组为单纯胰岛移植组，C组为胰岛和胸腺联合移植组，D组为胸腺细胞胸腺内预注射后，胰岛和胸腺联合移植组。移植后用酶联免疫吸附试验（ELISA法）检测IL－2、SIL－2R含量。结果：血清中IL－2在胰岛排斥前4d开始有显著升高（P＜0．001），而SIL－2R在排斥前3d开始显著升高（P＜0．001）。两者含量一直升高至排斥当日。结论：检测IL－2、SIL－2R含量在异种胰岛移植是否发生排斥反应具有重要意义。     

阿德福韦治疗肝移植术后乙肝病毒再感染的研究

目的 探讨和评价阿德福韦对因拉米呋啶耐药而出现HBV再感染的肝移植病例的抗病毒疗效。方法 9例存在拉米呋啶耐药株的肝移植术后HBV再感染者，治疗组（8例）予以改服阿德福韦酯，对照组（1例）加服泛昔洛韦治疗，并对两组病例的HBV DNA复制、乙肝血清学标志物、肝功、肾功等指标进行定期随访。结果 治疗组6例（75％）于服药后2～3个月均实现HBV DNA的阴转，1例（12．5％）维持低水平状态（10^3 copies／ml），且各有1例分别实现了HBsAg的阴转和HBeAg／HBeAb的血清学转换，肝功能均明显改善。对照组治疗前后各项指标无明显变化。结论 对拉米呋啶耐药而导致的HBV再感染的肝移植病人，新一代核苷类药物阿德福韦，能够有效的抑制HBV变异株的复制，改善肝功能。     

大鼠胰岛微囊化延长异种移植物在糖尿病小鼠体内生存期

本实验用胶原酶消化法分离新生大鼠胰岛，培养和Ｆｉｃｏｌｌ密度梯度离心纯化胰岛。用海藻酸－聚赖氨酸－海藻酸生物膜包裹胰岛并进行体外胰岛素分泌的能测定和体内异种移植实验。１０只链脲霉素诱导的糖尿病小鼠腹腔内移植微囊化胰岛３天后，小鼠血糖降至正常，体重逐渐增加，糖尿病得到纠正。维持最长者达１３０天，平均８５．２天。明显优于单纯胰岛植组，其功能维持不足９天。肾脏病理组织染色和透射电镜显示早期微囊化胰岛移植     

脂溢性脱发敏乐啶局部治疗临床观察

为评价敏乐啶溶液治疗脂溢性脱发的效果，作者进行了临床观察。选择２５０例脂溢性脱发患者进行研究，分为二组，敏乐啶组１８６例，用敏乐啶溶液治疗；对比组６４例，用３％樟脑酊治疗。患者均为男性，年龄２６～６５岁，病程１～２６年。两组患者年龄、病程及脱发类型在统计学上无显著性差异。嘱患者每日搽药两次，共观察６个月。敏乐啶组１８６例中，基本痊愈５例，显效７６例，好转７８例，无效２７例，瘙痒消失１１２例，总有效率为８５．５％，止痒率６０．２％。对比组６４例中，无一例基本痊愈，显效９例，好转２５例，无效３０例，瘙痒消失３１例，总有效率５３．１％，止痒率４８．４％。不良反应发生率敏乐啶组１．１％，对比组４．７％，表现为轻度皮肤红斑及瘙痒。敏乐定溶液是治疗脂溢性脱发的有效药物，其疗效高于樟脑酊。     

大鼠和猪胰岛分离,纯化,包埋的研究

用胶原酶消化分离大鼠，猪胰岛，用葡聚糖或聚蔗糖梯度密度离心纯化胰岛并将包埋于海藻酸钠－聚赖氨酸－海藻酸钠微囊内，用双硫腙染色鉴别胰岛，用荧光染色计数胰岛存活率，将此法制备的微囊胰岛用于治疗Ｉ型糖尿病有良好的应用前景。     

联合使用库司托啶与异搏定对大鼠肝脏低温保存的作用

目的 研究库司托啶（Ｃｕｓｔｏｄｉｏｌ）与异搏定（Ｖｅｒａｐａｍｉｌ）联合应用对低温保存大鼠肝脏的保护作用。方法 将４０只Ｗｉｓｔａｒ大白鼠制作肝离体灌注模型,库司托啶和异搏定作肝脏灌注并置于４℃保存６、２４和４８ｈ后观察肝窦内皮细胞死亡率,肝功能以及肝细胞形态的的变化。结果 库司托啶和异搏定灌注组明显优于对照组,保存２４ｈ,库司托啶＋异搏定（ＣＶＡ）组,库司托啶组的肝窦内皮细胞死亡率分别为３０．     

大鼠和猪胰岛分离、纯化、包埋的研究

用胶原酶消化分离大鼠、猪胰岛，用葡聚糖或聚蔗糖梯度密度离心纯化胰岛并将其包埋于海藻酸钠－聚赖氨酸－海藻酸钠微囊内，用双硫腙染色鉴别胰岛，用荧光染色计数胰岛存活率。将此法制备的微囊胰岛用于治疗Ⅰ型糖尿病有良好的应用前景。     

右旋美托咪啶复合丙泊酚用于无痛肠镜的临床观察

目的探讨右旋美托咪啶复合丙泊酚用于无痛肠镜的临床效果及可行性。方法40例结肠镜检查患者，随机分为右旋美托咪啶复合丙泊酚组（Ⅰ组）及单纯丙泊酚组（Ⅱ组）各20例，观察并比较2组用药情况、循环不良反应、体动反应情况、清醒时间。结果2组SBP、DBP、RR、HR比较差异无统计学意义，Ⅰ组丙泊酚用量及体动反应均少于Ⅱ组，清醒时间比较差异无统计学意义。结论右旋美托咪啶复合丙泊酚用于肠镜的检查优于单纯用丙泊酚。     

扶芳藤含药血清对大鼠胰岛细胞的保护机制

目的  探讨扶芳藤含药血清对大鼠胰岛细胞的保护作用及其机制。方法  将40只雄性SD大鼠随机分为5组,每组8只,包括对照组（正常大鼠胰岛细胞予正常大鼠血清培养）、缺血预处理组（获取胰腺前阻断腹主动脉再开放,胰岛细胞予正常大鼠血清培养）、扶芳藤治疗组（正常大鼠胰岛细胞予大鼠扶芳藤含药血清培养）、扶芳藤组和空白组（正常大鼠分别予扶芳藤提取物或蒸馏水灌胃,用于制备大鼠血清）。采用双硫腙（DTZ）染色法观察并计算胰岛数量;采用吖啶橙（AO）/碘化丙啶（PI）染色法,计算胰岛细胞存活率;采用胰岛素释放实验计算刺激指数（SI）来评价胰岛细胞功能;采用谷胱甘肽（GSH）及一氧化氮（NO）试剂盒测定胰岛细胞内GSH、NO含量;采用逆转录聚合酶链反应（RT-PCR）法检测诱导型一氧化氮合酶（iNOS）信使核糖核酸（mRNA）的表达水平。结果  经DTZ染色后胰岛细胞呈特异性猩红色,各组间胰岛细胞数量比较,差异无统计学意义（均为P > 0.05）。随培养时间延长,各组胰岛细胞活性均逐渐降低,分离培养72 h后,与对照组比较,扶芳藤治疗组细胞存活率更高,差异有统计学意义（P < 0.05）。胰岛素释放实验检测结果提示,与对照组相比,缺血预处理组及扶芳藤治疗组的SI均升高,差异均有统计学意义（均为P < 0.05）。与对照组比较,缺血预处理组及扶芳藤治疗组胰岛细胞的GSH含量均升高、NO含量均降低、iNOS mRNA表达水平均降低,差异均有统计学意义（均为P < 0.05）。结论  扶芳藤能通过提高GSH、降低iNOS表达及NO产生,提高胰岛细胞存活率,增强胰岛功能。     

骨髓间充质干细胞对同种异体胰岛的保护作用

目的 研究小鼠骨髓间充质干细胞(mesenehymal stem cells,MSCs)对同种异体胰岛的保护作用.方法 将C57BL/6小鼠骨髓间充质干细胞按照3×104/孔的密度预先接种至24孔培养板,次日分离纯化BALB/c小鼠胰岛,将其分为链脲菌素(streptozotocin,STZ)处理(诱导化学损伤)、混合淋巴细胞反应(mixed lymphocyte reaction,MLR)处理(诱导免疫损伤)、空白处理三组,与预先接种的MSCs进行共培养.作为实验对照,另设三组胰岛在不加MSCs的条件下进行体外培养.5 d后,用吖啶橙(acridine orange,AO)/碘化丙啶(propidium iodide,PI)荧光染色评估胰岛活力,葡萄糖刺激的胰岛素分泌实验检测其功能,比较MSCs共培养组与对照组胰岛在低糖、高糖刺激下的胰岛素分泌量及刺激指数(即高糖刺激胰岛素分泌量/低糖刺激胰岛素分泌量比值).结果 AO/PI染色显示,STZ或MLR处理的胰岛中有大量红色荧光的死细胞,而与MSCs共培养的胰岛中死细胞明显减少,绿色荧光的活细胞明显增加;STZ或MLR处理组胰岛在低糖、高糖刺激下的胰岛素分泌水平及刺激指数均显著下降,而与MSCs共培养者较相应对照组胰岛功能明显改善(P＜0.05).结论 小鼠骨髓间充质干细胞可减轻同种异体胰岛的化学及免疫损伤,保护游离胰岛的活力与功能.

Abstract：

Objective To study the protection of mouse bone marrow-derived mesenchymal stem cells ( MSCs) for allogenic islets. Method MSCs from C57BL/6 mice were preceded to a 24-well culture plate with the density of 3 × 104/well. On the second day, islets were isolated, purified and divided to undergo streptozotocin (STZ) induced chemical injury and mixed lymphocyte reaction ( MLR) respectively. Then, treated and control islets were respectively divided into the following groups: islets + MSCs, STZ-islets + MSCs, and MLR-islets + MSCs. As control groups for their counterparts, treated or non-treated islets were also cultured without MSCs. On the 5th day incubation, glucose-stimulated insulin secretion test was performed to assess the function of islets in different groups, comparing their insulin-secretion amount stimulated by low or high glucose and the stimulation index determined by the ratio of (insulin amount secreted under high-glucose stimulation)/(insulin amount secreted under low-glucose stimulation ). Islet viability was evaluated by acridine orange (AO)/propidium iodide (PI) fluorescence staining. Result As shown by AO/PI staining, large numbers of dead cell with red fluorescence could be observed in STZ- or MLR- treated islets without MSCs, while the number of dead cells obviously reduced in MSC-cocultured islets with increased viable cells of green fluorescence. STZ- or MLR- treated islets exhibited apparently decreased insulin-secretion amount either under low- or high-glucose stimulation, as well as the stimulation index. The insulin-secretion function was significantly improved in islets cocultured with allogenic MSCs (P ＜ 0. 05 ). Conclusions Bone marrow-derived MSCs can protect isolated allogenic islets against chemical and immunological injury.     

Improved Islet Survival and Funtion With Rat Endothelial Cells In Vitro Co-Culture

Objective

Pancreatic islet transplantation is an emerging therapy for type 1 diabetes. To preserve its function, transplanted islets must be revascularized because arterial and venous connections are disrupted during islet isolation. The current paradigm is that islet revascularization originates from the transplant recipient. This study was designed to test whether the function of isolated islets can be retained by co-culture with thoracic aorta endothelial cells in vitro.

Methods

Sprague-Dawley rats were used in this study. The endothelial cells (ECs) were isolated from the thoracic aorta. The viability of the isolated islets was assessed by acridine orange/propidium iodide (AO/PI) double staining. The islets were either placed in standard cultures (group A) or in co-cultures with ECs (group B). Islet viablity was assessed by an insulin release assay.

Results

The islets in group B exhibited normal morphology with >90% staining positive as detected by AO/PI with 7 days. Insulin release assays showed a significantly higher simulation index (SI) in group B compared with group A (P < .05) except on the first day.

Conclusion

This study suggested that co-cultrue of freshly isolated rat islets with ECs improves postculture survival and islet function in vitro.     

db/db小鼠胰岛的分离与特征分析

目的 分离db/db小鼠胰岛,并对其特征进行检测和分析。方法 采用胶原酶V对10只db/db小鼠胰岛分离进行逆行胰管灌注和消化,用Ficoll-1077和Ficoll-1119进行胰岛的不连续密度梯度纯化,对分离的胰岛进一步进行手工挑选,并用DTZ进行胰岛和纯度鉴定,用透射电镜观察胰岛内部分泌颗粒等情况。结果 经本方法分离可得到db/db小鼠胰岛数量为（122.4±6.6）个/只,当量为（483.6±82.3）IEQ/只,与ICR小鼠胰岛分离结果差异有统计学意义（P<0.05）;db/db小鼠胰岛DTZ染色后显淡红色;胰岛大小指数为（3.96±0.64）,显著大于ICR小鼠胰岛（P<0.05）;透射电镜下显示β细胞中的胰岛素分泌颗粒减少,分泌颗粒颜色较浅。结论 采用本方法可分离得到db/db小鼠胰岛,为后续开展基于胰岛功能和特征变化的2型糖尿病患者治疗研究提供一种参考。     

Neonatal pig pancreatic duct-derived insulin-producing cells: preliminary in vitro studies

Neonatal pig pancreata could represent an ideal tissue resource for donor islets for transplantation trials. Because functional islet beta-cells could derive from precursors situated in the ductal system, and neonatal animals are better suitable than adults for recovering such elements, we have examined whether isolated neonatal pancreatic ducts (NPD) could form insulin-producing cells. NPD, retrieved from the pancreas by collagenase digestion, were cultured for 2 weeks. A compact tissue monolayer detached by trypsin was re-incubated to form upon culture. The primary tissue monolayer was plated, yielding secondary monolayers that were supplemented in culture with the following factors: insulin transferrin selenium, niacinamide, keratinocyte growth factor, and high glucose, which promoted formation of islet cell-like clusters during 30 days of culture. Upon reaching 50 to 100 microm in diameter, the cell clusters were subjected to morphologic examination (assessment of viability by staining with ethidium bromide+fluorescein diacetate [EB+FD]; staining for insulin with diphenylthiocarbazone [DTZ]); DNA assay; insulin radioimmunoassay both in the basal state and after in vitro static incubation with high glucose; immunolabeling with anti-insulin fluorescent antibodies. Of the cell clusters, 80% were composed of viable cells that faintly showed DTZ staining. Basal insulin was 16.7 microU/mL, but no insulin response was elicited by stimulation with high glucose. Acid-ethanol extraction showed high insulin levels in the clusters. Finally, immunofluorescence for insulin was positive, indicating the presence of beta-cell-like committed elements. In conclusion, NPD may differentiate into insulin-producing cells, which are at a very early stage when the glucose-sensing apparatus is still immature.     

Variation in human islet viability based on different membrane integrity stains

Membrane integrity fluorescent staining is used routinely to evaluate islet viability. Results are used as one of the determining factors in islet product release criteria, and are used to assess the efficacy of different culture conditions. Recently, it has been observed that there is variation in the viability staining of freshly isolated islets based on which viability assay is used. This investigation compares three membrane integrity stains for the viability assessment of isolated human islets. Fluorescein diacetate/propidium iodide (FDA/ PI), the current standard method for assessing islet viability, demonstrates intense extracellular fluorescence, reducing the differential staining of intact islets. We further evaluated SYTO-13/ethidium bromide (SYTO/ EB) and calcein AM/ethidium homodimer (C/EthD) as alternative viability assays, and found considerable variation between FDA/PI and either SYTO/EB or C/EthD staining. Preparations of human islets were obtained from cadaveric pancreata after collagenase digestion, mechanical separation, and purification by continuous Ficoll gradient centrifugation. For each preparation, two replicate samples of 50 islets were counted for each stain, and the percent viability calculated. The results for SYTO/EB and C/EthD were nearly identical [57.6 +/- 7.3% and 57.9 +/- 7.2%, respectively (mean +/- SEM), N = 11]. FDA/PI-stained islets, however, showed consistently elevated values when compared to SYTO/EB. Accurate assessment of islet viability remains a critical determinant of islet product release. The discrepancies found between FDA/PI scoring and visual quality, compared with alternative stains, suggests that the FDA/PI stain may not be the optimal approach to assess islet viability.     

Experimental islet isolation in porcine pancreas with new enzyme Liberase PI

The aim of this study was to investigate the results of 20 consecutive porcine islet isolations using a new enzyme Liberase PI. Twenty pancreata were procured for islet isolation, which was performed using modified Ricordi's method with Liberase PI. Quantitation of islet viability staining, insulin stimulation assay, intracellular insulin content/DNA, and in vivo transplantability into diabetic nude mice were examined for quality control. The results were compared between a high-yield group (>2500 IEQ/g pancreas) and a low-yield group (<2500 IEQ/g pancreas). Sufficient amount of purified islets (3000 IEQ/g pancreas) were obtained using the new brand enzyme Liberase PI. These islets showed good quality in structure and functions, which were demonstrated by in vitro and in vivo standard assays. Isolation index (IEQ/number) of the low-yield group was lower than that of high-yield group (0.75 vs 0.86), which means more fragmentation of islets in the low-yield group. There were no differences in function between the two groups. In conclusion, we obtained sufficient numbers of viable, functional islets from porcine pancreas using a new brand enzyme Liberase PI and low-temperature isolation technique. However, overdigestion of islets during the isolation remains to be overcome. Advance in porcine islet isolation technique will in the future make the porcine islet xenotransplantation a reality for the cure of diabetes mellitus.     

一种经济高效的SD大鼠胰岛细胞分离技术

目的 建立一种经济高效的大鼠胰岛细胞分离纯化方法,为胰腺的修复重建奠定实验基础.方法 成年雄性SD大鼠25只,体重230～380g,共进行5次实验,每5只大鼠一组进行消化和分离.采用医用复方氯化钠注射液(compound sodium chloride injection, CSCI)经胰总管灌注大鼠胰腺,0.5mg/mL V型胶原酶消化后,分别采用浓度为27.0%、23.0%、20.5%和11.0%的Ficoll 400形成不连续密度梯度介质,离心纯化胰岛细胞.双硫腙(dithizon, DTZ)染色行纯化前后胰岛细胞计数和纯度检测;荧光染料碘化丙啶(propidium iodide, PI)和二乙酸荧光素(fluorescein diacetate, FDA)储存液双染色鉴定胰岛细胞活性;RPMIl640培养基培养3d后,分别用浓度为2.8mmol/L的低糖和25.0mmol/L的高糖行葡萄糖刺激胰岛素释放实验检测胰岛细胞功能.结果 5次实验胰岛细胞消化时间为(13.8±1.6)min.DTZ染色鉴定纯化前胰岛细胞数为(5626±422)个,纯化后为(2914±485)个,纯化后的胰岛细胞数较纯化前明显减少(P<0.01),回收率51.6%±6.0%,每个胰腺收获胰岛细胞数为(583±97)个/只.5次分离获得的胰岛细胞纯度为90.2%±3.4%,活性为81.6%±7.0%.培养3d后,葡萄糖刺激胰岛素释放实验显示:低糖环境下胰岛素水平为(39.7±7.5)EU/L,高糖环境为(116.1±17.4)EU/L,比较差异有统计学意义(P<0.01)刺激指数为3.0±0.4.结论 采用CSCI作为大鼠胰岛细胞分离纯化的主要液体试剂,并采用低浓度V型胶原酶消化,不仅可降低实验成本,同时可获得高质量的胰岛细胞.     

In vitro staining of islets of Langerhans for fluorescence-activated cell sorting.

A previously described technique from the author's laboratories for purification of pancreatic islets by fluorescence-activated cell sorting used the dye neutral red (NR) to obtain specific fluorescence of islets sufficient to give a sorting signal. A major drawback with this technique was the need to inject the dye intravascularly before excision of the pancreas. Preliminary investigations showed that NR would produce selective staining of islets by topical application in vitro but only at low concentrations that were insufficient to give fluorescence strong enough for sorting. The chelating agent dithizone (DTZ) produces bright red staining of islets by topical application in vitro. Further studies showed that dithizone-stained islets exhibited moderately strong fluorescence that faded too quickly for reliable sorting. By combining both NR and DTZ staining in vitro, selective fluorescence of islets was obtained that was sufficient to allow efficient sorting. Using the combined DTZ/NR stain the yield of islets obtained by sorting from a single rat pancreas was 569 +/- 72 (n = 16), corresponding to 83% of the islets present in the digest. The mean purity of the preparation, confirmed by histologic examination, was 80%. The viability of the islets was shown to be good both by supravital staining and by the successful correction of streptozotocin diabetes in syngeneic rats following transplantation of sorted islets.     

Establishment of fluorescein diacetate and ethidium bromide (FDAEB) assay for quality assessment of isolated islets

One of the most important factors in clinical islet transplantation is isolation of a great number of islets with good viability. According to viability assessments of isolated islets, the static incubation test and the perifusion test of islets, which are used retrospectively, take much time and need various apparatus. But viability assessments of isolated islets for clinical islet transplantation require a simple, rapid, sensitive, and prospective method. We have developed a microfluorometric viability assay for isolated human, porcine, and dog islets of pancreata using fluorescein diacetate and ethidium bromide (FDAEB). Fluorescein diacetate (FDA) causes live cells to fluoresce green under blue light excitation (490 nm) and ethidium bromide (EB) causes dead cells to fluoresce red. In this study, we investigated the applicability of FDAEB staining to quality assessment of isolated islets for clinical use by correlation with the counting method with insulin secretion of islets. Discrimination of living from dead islets by insulin secretion correlated well with viability as determined by FDAEB staining. The proportion of living islets within isolated canine islets, as measured by microfluorometric counting, was found to correlate highly significantly on low-temperature (24 degrees C) culture (R = 0.831, p < 0.001) and on 37 degrees C culture (R = 0.553, p < 0.05) with the insulin contents of the same islets. Therefore, it is possible to differentiate degrees of viability, and a scoring system is described for this purpose. The FDAEB assay prospectively and easily provides a rapid, accurate, and objective measurement of the proportion of living cells and dead cells in isolated islets for clinical islet transplantation.