【作者】 赵芳；

【导师】 朱旅云；

【作者基本信息】 河北医科大学 ， 内科学， 2010， 硕士

【摘要】 目的:前期实验利用胰岛细胞生存的微环境已经将人脐带Wharton’s Jelly中间充质干细胞(hUC-MSCs)诱导分化为具有胰岛素分泌功能的胰岛样细胞,但是这些诱导分化的胰岛样细胞在体内的功能如何尚不确定。本实验旨在观察hUC-MSCs诱导分化的胰岛样细胞对1型糖尿病大鼠血糖及体重的影响。方法:1采用组织块贴壁法分离培养hUC-MSCs ;2利用胶原酶ⅴ分次消化大鼠胰岛细胞;3应用Transwell板将hUC-MSCs与大鼠胰岛细胞共培养,利用胰岛细胞生存的微环境将hUC-MSCs向胰岛样细胞诱导分化,倒置显微镜下观察细胞形态变化;4将SD大鼠随机分为正常对照组和糖尿病模型组。腹腔注射链脲佐菌素(STZ)制作1型糖尿病大鼠模型,随机血糖连续3次>16.7mmol/L定为糖尿病大鼠造模成功。将造模成功的糖尿病大鼠随机分为两组:STZ实验组(糖尿病大鼠肾被膜下移植诱导后的胰岛样细胞)和STZ对照组(糖尿病大鼠不做任何处理);5在移植前应用Brdu对诱导后的胰岛样细胞进行标记;6将Brdu标记后的胰岛样细胞移植到STZ实验组糖尿病大鼠的肾被膜下,观察大鼠血糖、体重的变化,8周后结束实验。7实验结束时将大鼠的胰腺和肾脏摘除,胰腺行HE染色,肾脏行HE染色和免疫组化染色。结果:1腹腔注射STZ后大鼠的血糖明显升高,出现多尿、多饮、体重增加缓慢甚至降低,皮毛颜色暗淡,有脱毛现象。随机血糖连续3次>16.7mmol/L定为糖尿病大鼠造模成功,造模成功率为100%。2在250倍镜下随机选取10个视野计数阳性细胞数与非阳性细胞数,计算Brdu阳性率=阳性细胞数/(阳性细胞数+非阳性细胞数),得出Brdu的标记率为92%。3在实验过程中,STZ实验组有5只大鼠死亡(2只死于手术,3只死于高血糖),STZ对照组有8只大鼠死亡(均死于高血糖),最后进行统计分析的共17只(正常对照组6只,STZ实验组7只,STZ对照组4只)。4大鼠血糖的变化细胞移植后8周,STZ实验组的血糖较移植前下降,差异具有统计学意义(p<0.05)。STZ对照组和正常对照组的血糖与移植前比较无明显差异(p>0.05)。STZ实验组的血糖移植前为(29.00±3.68)mmol/L,移植后为(20.31±1.70)mmol/L;STZ对照组的血糖实验前为(27.87±2.28)mmol/L,实验后为(28.23±1.89)mmol/L;正常对照组的血糖实验前为(6.70±0.25)mmol/L,实验后为(6.58±0.66)mmol/L。5大鼠体重的变化细胞移植后8周,三组大鼠的体重较移植前均增加,差异均具有统计学意义(p<0.05)。正常对照组增加的最多,STZ实验组次之,STZ对照组增加的最少,差异具有统计学意义(p<0.05)。STZ实验组的体重移植前为(219.33±16.58)g,移植后为(323.14±28.33)g;STZ对照组的体重实验前为(207.25±13.63)g,实验后为(246.00±27.54)g;正常对照组的体重实验前为(275.33±28.49)g,实验后为(496.50±20.51)g。6胰腺HE染色示:正常对照组大鼠的胰腺内散在分布大小不等、形状规则的胰岛,胰岛的结构清晰。STZ对照组糖尿病大鼠的胰岛数量明显减少,形状不规则,萎缩胰岛周围腺泡细胞肥大。胰腺间质大动脉收缩状,内皮细胞突向管腔,动脉壁增厚,动脉周围纤维组织增生,可见神经纤维束。胰腺小叶萎缩,腺泡腔明显,间质淋巴细胞浸润。STZ实验组大鼠的胰腺组织与STZ对照组有相似的改变,无自发恢复现象。7肾脏HE染色示:正常对照组和STZ对照组大鼠的肾被膜边缘完整,形态清楚。STZ实验组大鼠在移植细胞的部位肾被膜和肾皮质之间有大量的细胞,部分肾被膜出现断裂现象。8肾脏免疫组化染色示:正常对照组和STZ对照组大鼠的肾被膜下无胰岛素、Brdu染色阳性的细胞。STZ实验组大鼠在移植细胞的部位肾被膜和肾皮质之间有胰岛素、Brdu阳性的细胞。结论:人脐带Wharton’s Jelly中的间充质干细胞与大鼠胰岛细胞共培养诱导分化的胰岛样细胞移植到糖尿病大鼠肾被膜下可在局部存活,并且能够使大鼠的血糖降低、体重增加。更多还原

【Abstract】 OBJECTIVE: Mesenchymal stem cells in Wharton’s Jelly of the human umbilical cord (hUC-MSCs) had been transdifferentiated into islet-like cells utilizing the microenvironment that islet cells living on the protophase. But it was unknown that if these islet-like cells could react in vivo. This study was to explore the effect of these islet-like cells on the levels of blood glucose and body weight in rats with type 1 Diabetic Mellitus (DM).METHODS:1 MSCs were isolated and cultured from Wharton’s Jelly of the human umbilical cord by tissue adherence.2 The islet cells were isolated from pancreas by collagenase V digesting.3 hUC-MSCs were induced by cocultureing with islet cells. Cell morphologic change was observed under inverted microscope.4 Rats were divided into normal control group and diabetic model group. The models of type 1 DM were made by intraperitoneal injection streptozocin(STZ). If random blood sugar of rats exceeded 16.7mmol/L for continuous three times, diabetic models were considered to succeed. The diabetic rats were divided into STZ experiment group and STZ control group.5 Postinduced cells were marked by Brdu.6 The positive cells for Brdu were transplanted into renal capsule of diabetic rats in STZ experiment group. Blood glucose and body weight of diabetic rats were observed.The experiment was ended after 8 weeks.7 The pancreas and kidney were removaled to carry out HE or immunohistochemistry dyeing at the end of the experiment.RESULTS:1 Blood glucose of rats increased remarkably after intraperitoneal injection STZ. Rats occurred urorrhagia and polydipsia symptoms. Their body weight didn’t increase even cut down. The fur of diabetic rats were dim and some fur depilated. If random blood sugar of rats exceeded 16.7mmol/L for continuous three times, diabetic models were considered to succeed. The achievement ratio of diabetic models was 100 percent.2 Selecting ten visual fields randomly to count positive cells and negative cells under the microscope. Then, to calculate the positive rate of Brdu. The positive rate of Brdu was 92 percent.3 In the process of experiment, five rats died in STZ experiment group, two of which died of operation and three of which died of hyperglycemia. Eight rats died of hyperglycemia in STZ control group. There only had seventeen rats to carry out statistical analysis eventually.4 The change of blood glucose At eight weeks after cell transplantation, blood glucose in STZ experiment group decreased, there had statistical significance(p<0.05). Blood glucose in STZ control group and normal control group all had no statistical significance(p>0.05). Blood glucose in STZ experiment group were (29.00±3.68) mmol/L before cells transplantation and (20.31±1.70) mmol/L after cells transplantation. Blood glucose in STZ control group were (27.87±2.28) mmol/L before cells transplantation and (28.23±1.89) mmol/L after cells transplantation. Blood glucose in normal control group were (6.70±0.25) mmol/L before cells transplantation and (6.58±0.66) mmol/L after cells transplantation.5 The change of body weight At eight weeks after cell transplantation, body weight increased in three groups, there all had statistical significance (p<0.05). The increment of body weight in normal control group was most, the second was STZ experiment group. The increment of body weight in STZ control group was least. Blood glucose in normal control group were (275.33±28.49) g before cells transplantation and (496.50±20.51) g after cells transplantation. Body weight in STZ experiment group were (219.33±16.58) g before cells transplantation and (323.14±28.33) g after cells transplantation. Blood glucose in STZ control group were (207.25±13.63) g before cells transplantation and (246.00±27.54) g after cells transplantation.6 The HE dyeing showed that inequality of size pancreatic islet diffused distribution in normal pancreas. Their structures were clear. The quantity of islet decreased in diabetic rats of STZ control group. Their shapes were rugosity. Some hypertrophic acinar cells surrounded emarcide islets. Some large arteries were contractus in the interstitium of pancreas. Endothelial cells faced to lumens and the artery walls were incrassate. The fibrous tissue surrounding artery hyperplasy and formed some nerve fiber bundles. The lobule of pancreas atrophy. There had evident acinar lumina and some lymphocytes infiltrate into interstitium. The pancreas in STZ experiment group had the similar change to STZ control group.7 The HE dyeing showed that the verge of renal capsule was integrity and clear in normal control group and STZ control group. There had considerable cells between renal capsule and renal cortex in STZ experiment group. The renal capsule appeared break.8 The immunohistochemistry dyeing showed that there didn’t have positive cells for insulin or Brdu below the renal capsule in normal control group and STZ control group. There had some positive cells for insulin whose endochylemas were brown as well as some positive cells for Brdu whose nucelus were brown between renal capsule and renal cortex in STZ experiment group.CONCLUSION: Islet-like cells derived from mesenchymal stem cells in Wharton’s Jelly of the human umbilical cord could survival below the renal capsule of diabetic rats. These islet-like cells could lower blood glucose and increase body weight after cell transplantation.更多还原