本文選題：胰十二指腸同源盒基因-1　切入點(diǎn)：人脂肪間充質(zhì)干細(xì)胞　出處：《青島大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的探討胰十二指腸同源盒基因-1(PDX-1)在人脂肪間充質(zhì)干細(xì)胞(h ADSC)分化為胰島分泌細(xì)胞中的作用。方法將凍存的人脂肪間充質(zhì)干細(xì)胞進(jìn)行復(fù)蘇,并隨機(jī)分為實(shí)驗(yàn)組與對(duì)照組,實(shí)驗(yàn)組:將攜帶胰十二指腸同源盒基因-1的病毒液(MOI(感染復(fù)數(shù))=50)加入細(xì)胞懸液中,混合培養(yǎng)4小時(shí)后,去除含有病毒的培養(yǎng)液,更換新鮮的完全培養(yǎng)液,繼續(xù)培養(yǎng)至48小時(shí),以保證轉(zhuǎn)染效果;對(duì)照組:采用不含病毒液的培養(yǎng)液培養(yǎng)48小時(shí)。然后分別采用蛋白質(zhì)印跡法(Western-blotting)和細(xì)胞免疫熒光染色方法對(duì)感染結(jié)果進(jìn)行檢測。對(duì)感染后15天細(xì)胞利用雙硫腙(DTZ)染色觀察胰島素陽性反應(yīng)細(xì)胞,酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測細(xì)胞胰島素的分泌量,細(xì)胞免疫熒光染色法檢測胰島分泌相關(guān)基因insulin 1的表達(dá)。結(jié)果(1)實(shí)驗(yàn)組的人脂肪間充質(zhì)干細(xì)胞進(jìn)行病毒感染48h后,Western blot法和免疫熒光法均可檢測到PDX-1的表達(dá),對(duì)照組的細(xì)胞則無PDX-1的表達(dá),并且感染的細(xì)胞在15d之后仍能檢測到PDX-1的表達(dá)。(2)實(shí)驗(yàn)組出現(xiàn)胰島素陽性細(xì)胞且被染成棕紅色,對(duì)照組未出現(xiàn)胰島素陽性細(xì)胞。(3)感染后15d,實(shí)驗(yàn)組和對(duì)照組細(xì)胞在低糖環(huán)境下分泌的胰島素量分別為(6.98±0.72)、(6.78±0.54)ng/m L,兩組比較,P0.05,無統(tǒng)計(jì)學(xué)意義;高糖環(huán)境下分泌的胰島素量分別為(13.38±0.66)、(6.50±0.64)ng/m L,兩組比較,P0.05,有統(tǒng)計(jì)學(xué)意義;實(shí)驗(yàn)組胰島分泌相關(guān)基因insulin 1的表達(dá)量多于對(duì)照組。結(jié)論胰十二指腸同源盒基因-1在人脂肪間充質(zhì)干細(xì)胞向胰島分泌細(xì)胞分化過程中具有促進(jìn)作用,對(duì)開展胰島移植治療I型糖尿病具有重要意義。
[Abstract]:Objective to investigate the role of pancreaticoduodenal homologous box gene-1 PDX-1 in differentiation of human adipose mesenchymal stem cells into pancreatic islet secretory cells. Methods cryopreserved human adipose mesenchymal stem cells were resuscitated and randomly divided into experimental group and control group. In the experimental group, the virus moi containing the gene 1 of pancreaticoduodenal homologous box 1 was added to the cell suspension. After 4 hours of mixed culture, the virus-containing culture medium was removed, the fresh complete culture medium was replaced, and the culture was continued for 48 hours. To ensure the transfection effect; The control group was cultured in virus-free medium for 48 hours. Then the infection results were detected by Western blotting and cell immunofluorescence staining. The cells were stained with dithizone DTZ 15 days after infection. Color observation of insulin positive reaction cells, Enzyme linked immunosorbent assay (Elisa) was used to detect the secretion of insulin. The expression of human adipose-derived mesenchymal stem cells (HMSCs) in the experimental group was detected by Western blot assay and immunofluorescence assay after 48 h of viral infection. The cells in the control group showed no PDX-1 expression, and the infected cells could still detect the expression of PDX-1 after 15 days.) Insulin positive cells were found in the experimental group and were stained brown red. In the control group, the amount of insulin secreted by the cells of the experimental group and the control group was 6.98 鹵0.72 鹵0.54 ng / mL at 15 days after infection. There was no significant difference between the two groups (P 0.05). The amount of insulin secreted in high glucose environment was 6.50 鹵0.64 ng / mL, respectively, which was significantly higher than that in the control group (P 0.05). The expression of pancreatic islet secretory gene insulin 1 in the experimental group was higher than that in the control group. Conclusion the pancreaticoduodenal homologous box gene 1 plays an important role in the differentiation of human adipose mesenchymal stem cells into pancreatic islet secretory cells. It is of great significance to carry out islet transplantation in the treatment of type I diabetes.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.1

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