目的探讨重组人碱性成纤维生长因子（rhbFGF）基因导入胰岛细胞团后经肾被膜下移植,对移植物微血管重建的影响。方法采用携带rhbFGF-flag-GFP基因的腺病毒载体体外转染培养的胰岛细胞团,再移植至同系糖尿病小鼠的肾被膜下,术后30 d内观察糖尿病治疗情况,通过术后14 d糖耐量试验和HE、免疫组化等观察胰岛功能和移植物的微血管重建结果。结果感染复数（MOI）为100的rhbFGF基因转染后胰岛与未处理的胰岛在体外培养60 h后活性无统计学差异（P=0.75）;移植200 IEQ转基因胰岛至体内14 d后,胰岛功能良好,微血管重建增加,优于单独400 IEQ胰岛移植组。结论 rhbFGF基因导入胰岛移植有助于提高胰岛的功能,促进移植物微血管重建,并可减少胰岛移植量。

目的:制备得到小鼠抗家兔胰岛细胞的多克隆抗体.方法:选择雄性新西兰大白兔作为胰岛供体,采用胆总管灌注胶原酶的方法进行胰岛细胞团的分离,用磷酸盐缓冲液洗涤细胞,加入弗氏完全佐剂或弗氏不完全佐剂超声乳化后,选择雌性Balb/c小鼠进行多次免疫接种,收集含抗体的小鼠血清,进行家兔胰腺组织的免疫组化实验,观察阳性染色情况.结果:通过家兔胰腺组织HE染色和免疫组化染色,可明显观察到胰岛部位呈阳性染色,确认小鼠血清中存在抗家兔胰岛细胞多克隆抗体.结论:通过本方法可制得小鼠抗家兔胰岛细胞多克隆抗体.

Islet transplantation is one of the clinically applied methods in the treatment of diabetes. However, there are still some problems, such as the insufficient of donors, the slow and insufficient reconstruction of microvascular in grafts, and so on. In this study, plasmids containing recombinant human fibroblast growth factor (rhbFGF) gene were introduced into islets by adenovirus vectors in vitro, and exploring the appropriate multiplicity of infection (MOI). In vivo, islets infected by adenovirus-rhbFGF gene were transplanted into the renal capsule of homogeneously diabetic mice. In vitro, islet activity in different groups was evaluated by FDA-PI fluorescence staining, repairation of microvascular basement in islets after infection was observed by transmission electron microscopy, islet function was assayed using glucose stimulation and insulin release. 200 IEQ islets infected by Ad-rhbFGF-3* flag-GFP with appropriate MOI were transplanted into diabetic mice under the renal capsule. The blood glucose and the oral glucose tolerance test in 14 days after the transplantation were monitored, and the effect of microvascular reconstruction of the grafts transfected with rhbFGF gene was investigated. The results showed that the activity of islets infected by 100 MOI adenovirus-rhbFGF gene recovery significantly after 60 h of culturation, and the islet endothelial cells were significantly repaired after 36 and 60 hours of culture in this group. The results of glucose stimulation showed that the insulin release in this group was significantly higher than that in other groups follow the high glucose stimulation. Diabetic mice transplanted with 400 IEQ islets or 200 IEQ islets infected with 100 MOI adenovirus-rhbFGF gene in vivo, could maintain a normal blood glucose for a long time. OGTT results showed that there was no significant difference between the test group and normal mice. The study laid a good foundation for islet transplantation with reduced dosage and stable blood glucose control.

In this paper, we introduced a simple way to transplant islets to the kidney subcapsule in mice and then evaluated the function of the transplanted islet. The islets were harvested by reverse perfusion of collagenase V into the common bile duct and purification by Histopaque density gradient. After a simple culture, the islets were transplanted under the kidney capsule of diabetic Balb/c mice using a gel loading tip. Then, blood glucose and oral glucose tolerance were tested regularly. The kidney was cut on the 10th day after the islet transplantation. Hematoxylin-eosin (HE) and immunofluorescence staining of the islet under the kidney capsule were performed to evaluate the islet morphology and function, and transmission electron microscopy was performed to examine the ultrastructure of the islet grafts in the subcapsule. We obtained 789 253 islets or 865.6 124.6 I EQ from four mice in one isolation and infused 350-400 IEQ islets to a diabetic mouse in one transplant. The blood glucose returned to normal 24 h after the islet transplantation. The OGTT results showed that the blood glucose levels of mice after islet transplantation were significantly lower than those of mice with diabetes (p = 0.001). The immunofluorescence staining results showed that the activity of the islets was adequate. The peak blood glucose in OGTT was significantly lower in mice with transplanted islets than in diabetic mice (p = 0.001), and the insulin concentration of diabetic mice with islet transplantation increased significantly as compared to diabetic mice at 0, 20, and 60 min. However, the transmission electron microscope (TEM) result showed that the microvessels lacked endothelia in the islet grafts. This experiment creates a solid foundation for further research on drug screening for islet transplantation.

目的 介绍一种新的家兔胰岛分离纯化方法并进行纯化后胰岛的外观、活性和胰岛细胞团表面结构等质量分析.方法 雄性日本大耳白,采用2 mg/mL胶原酶Ⅴ逆行胰管灌注和消化,并用Hanks液洗涤,用Histopaque-1119(R)、Histopaque-1077(R)密度梯度离心进行胰岛纯化,DTZ、FD-PI染色鉴定胰岛纯度和活性,扫描电镜观察纯化后胰岛表面结构.结果 家免胰管开口于小肠,纯化得到家兔胰岛外形圆整,纯度＞90％,活性(87.4±5.9)％,纯化前胰岛当量为(1 874.8±464.5)IEQ,纯化后胰岛当量为(1 254.8±235.8)IEQ;扫描电镜结果显示胰岛细胞团表面有被膜,但部分胰岛被膜被消化.结论 采用此方法可快速得到纯度高、活性好且结构完整的家兔胰岛,为进一步进行家免胰岛体外质量及体内移植研究奠定了基础.

Objective:To develop a small type of suppository of immunosuppressive agents to suit for large animals after abdominal organ transplantation, and carry out it’s quality evaluation. Methods:Rapamycin and ta-crolimusas were chosed as active pharmaceutical ingredients, and polyethylene glycol 2000, polyethylene glycol 4000 were used as substrate. Melting followed with a quantitative injection molding process was used to prepare the small type of compound suppository. The appearance, weight difference, melting time and drug content of the suppository were evaluated. Results:The compound rapamycin suppository was translucent, white, and had good hardness at room temperature. It’s short, and the weight variation, melting time and drug content were con-formed to the requirements of the suppository. Conclusion:The compound rapamycin suppository prepared by this method conforms to the quality of suppository, which makes a good foundation for the development of the new drug.

Objective In this paper,we introduced a simple and easy way to do islet transplantation under kidney capsule in mouse,and the effect evaluation of it' s blood glucose,oral glucose tolerance and histological stain after transplantation.Methods BALB/c mice are used in the experiment.The islets were harvested by reversely perfusion of collagenase V into common bile duct and purification by Histopaque density gradient followed by manual picking.After a simple culture,the islets were transplanted under the kidney capsule of diabetic BALB/c mice using a gel loading tip.Then the blood glucose and the oral glucose tolerance test were tested regularly.The kidney was cut at the 10th day after the islet transplantation.The HE and immunofluorescence staining of islet under kidney capsule were also done to evaluate the islet morphology and function.Results We get 1 063 ± 122 islets,or 1 259.72 ± 107.19 IEQ from four mice at one time.The level of blood glusoce turned down to normal in 24 h after the islet transplantation.Oral glucose tolerance test (OGTY) results showed that the blood glucose levels of mice after islet transplantation were significantly lower than those of diabetes (P ＜ 0.01).And the results of immunofluorescence staining showed that the activity of islets was good [(92.8 ± 2.6) ％].Conclusion The method of the experiment is simple,and the islet activity is good after transplantation,which make a good foundation for the further research on drug screening for islet transplantation.

目的:制备一种含雷帕霉素的聚乙二醇栓剂,并对其质量进行评价。方法:以雷帕霉素为原料药,聚乙二醇2000和聚乙二醇4000为主要基质,热熔法工艺制备含药栓剂,并对其药物含量、重量差异、融变时限、药物溶出等进行评价。结果:雷帕霉素聚乙二醇栓剂外观呈白色半透明状,室温下保持较好的硬度,药物含量、重量差异和融变时限均符合栓剂要求,药物在释放介质中约25 min释放达到75%以上。结论:本研究制备的雷帕霉素聚乙二醇栓剂符合中国药典栓剂质量检测要求,有望开发成雷帕霉素的一种新剂型。

Ca-alginate-poly-l-lysine-alginate (APA-Ca) and Ba-alginate-poly-l-lysine-alginate (APA-Ba) microcapsules were prepared and their thickness and surface were examined by light microscopy and scanning electron microscopy. Specifically, light microscopy with frozen section was used to visualize and quantify the thickness of APA membrane, and monitor temporal changes in the thickness of microcapsules during a month long culture in vitro. The section graph of APA microcapsule represents the accurate measurement of layer thickness of APA-Ca with diameter 900 ± 100 and 500 ± 100 μm at 6.01 ± 1.02 and 9.54 ± 2.42 μm (p < 0.05), and layer thickness of APA-Ba with diameter 900 ± 100 and 500 ± 100 μm at 5.47 ± 0.90 and 8.21 ± 1.97 μm (p < 0.05), regardless of the alginate composition used to generate the microcapsules. The microcapsule was stable during the culture for 30 days in vitro. Field emission scanning electron microscopy with freeze drying method was used to detect the surface and thickness of dried microcapsules. From the results, the outer surface of APA-Ca and APA-Ba membrane were smooth and dense, the film thickness of the APA-Ca was about 450-690 nm, while the APA-Ba was approximately 335 nm. In vivo experiment, little significant difference was seen in the change of film thickness of microcapsules in intrapertioneal site for 30 days after transplantation (p > 0.05), except that the recovery of APA-Ba was higher than the APA-Ca microcapsules. The paper showed an easy method to prepare APA-Ca and APA-Ba, and examine their thickness and surface, which could be utilized to study other types of microcapsules.

Objective:To explore a visual methold of the membrane thickness of APA microcapsules and their surface structures,and determinate the film thickness and surface characteristics.Methods:We use methylene blue to dye the film of microcapsules,then within frozen section and optical microscope the the thickness of microcapsule were observed;and the thickness of microcapsules hatched in 37 ℃ for a month were also assayed.The surface of membrane were studied by ice emission scanning electron microscopy after lyophilization.Results:The member thickness of each APA microencapsules is not uniform.After incubated for 30 d in 37 ℃,there is no obviously difference in the thichness than befor(P>0.05).Surfaces of two microencapsules are smooth and compact under the scanning electron microscopy.The thickness of APA-Ca microcapsules are 450-690 nm,which of APA-Ba are about 350 nm.Conclusion:The essay provided a simple method on how to determine the membrane thickness and surface,which would be an important reference to expound the APA microcapsules.

目的:通过考察不同处方和工艺制备的吲哚美辛海藻酸钠-壳聚糖-海藻酸钠(ACA)微囊的体外释放,研究影响囊膜通透性及膜强度的因素。方法:以吲哚美辛为模型药物,采用不同的交联剂氯化钙、氯化钡,改变海藻酸钠、壳聚糖、柠檬酸钠浓度及制备工艺来制备微囊,以药物的体外释放曲线来评价囊膜的通透性,通过超声破碎评价微囊的强度。结果:影响微囊中药物释放的主要因素为处方中的交联剂,提高海藻酸钠、壳聚糖浓度、降低液化囊心时间,微囊通透性下降;氯化钙为交联剂制备的微囊强度较小。结论:通过处方和工艺调节有望得到通透性和膜强度适宜的ACA微囊。