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目的探讨血管内皮生长因子(VEGF)对大鼠的移植胰岛再血管化及对其存活率和功能的影响。方法构建质粒plRES2-EGFP/VEGF_(165),以脂质体法在体外转染大鼠(供者)的血管内皮细胞。流式细胞术检测转染效率;免疫组织化学染色检测VEGF的表达。将糖尿病大鼠(受者)随机分为3组,每组10只。对照组:于肾被膜下单纯移植供者的300当量胰岛:实验组和空白转染组除移植供者的300当量胰岛外,分别加入供者的1×10~6个转染了质粒plRES2 EGFP/VEGF_(165)的血管内皮细胞和未经转染的正常血管内皮细胞。移植后监测受者的血糖及血清胰岛素水平。术后第14天,取受者肾脏.行HE染色及Insulin-6、VEGF和CD34免疫组织化学染色。结果血管内皮细胞中VEGF_(165)的转染效率为13.06%。实验组供者的血管内皮细胞胞核和胞浆中均有VEGF表达。实验组受者于移植术后第3天血糖及胰岛素水平恢复正常;对照组和空白转染组虽有所改善,但未恢复到正常水平,与实验组相比,差异有统计学意义。实验组受者肾被膜下可见成团胰岛,Insulin-6免疫组织化学染色呈阳性,周围及内部有大量内皮细胞;VEGF_(165)及CD34免疫组织化学染色呈阳性。对照组和空白转染组肾被膜下的细胞团中心细胞较少,部分被纤维组织代替,内部仅有少量CD34染色阳性的内皮细胞;Insulin-6免疫组织化学染色仅有少量细胞染成棕黄色;VEGF_(165)免疫组织化学染色呈阴性。结论供者的胰岛与转染了VEGF_(165)的血管内皮细胞共同移植给受者可以促进移植胰岛的再血管化,提高其存活率,并使其功能恢复正常。
Objective To investigate the effects of vascular endothelial growth factor (VEGF) on the revascularization of islets in rats and its survival rate and function. Methods Plasmid plRES2-EGFP / VEGF_ (165) was constructed and transfected into vascular endothelial cells of rats (donor) by liposome in vitro. Transfection efficiency was detected by flow cytometry. The expression of VEGF was detected by immunohistochemical staining. Diabetic rats (recipients) were randomly divided into three groups of 10. Control group: 300-equivalent islets of transplanted donor under renal capsule alone: The experimental group and blank transfection group were treated with 1 × 10 ~ 6 plasmids transfected with plasmid plRES2 EGFP / VEGF_ (165) of vascular endothelial cells and untransfected normal vascular endothelial cells. After transplantation, the recipient’s blood glucose and serum insulin levels were monitored. On the 14th postoperative day, the kidneys were harvested and HE staining and Insulin-6, VEGF and CD34 immunohistochemical staining were performed. Results The transfection efficiency of VEGF_ (165) in vascular endothelial cells was 13.06%. The expression of VEGF in the nuclei and cytoplasm of the vascular endothelial cells of experimental group donors. The recipients of experimental group returned to normal levels of blood glucose and insulin on the third day after transplantation. The control group and blank transfection group improved, but did not return to normal level, which was significantly different from the experimental group. Insulin-6 immunohistochemical staining was positive, with a large number of endothelial cells around and inside; VEGF165 and CD34 were positive for immunohistochemistry staining. The control group and blank transfection group under the capsule of the renal capsule less central cells, some were replaced by fibrous tissue, internal only a small amount of CD34-positive endothelial cells; Insulin-6 immunohistochemical staining only a small amount of cells stained brown ; VEGF165 immunohistochemical staining was negative. Conclusion Co-transplantation of donor islets with vascular endothelial cells transfected with VEGF_ (165) can promote the revascularization of transplanted islets, increase their survival rate and restore their functions.