PSMB5对人骨髓间充质干细胞增殖能力的影响

发布时间：2018-06-01 14:41

  本文选题：PSMB5 + 蛋白酶体　； 参考：《山西医科大学》2014年硕士论文

【摘要】：目的：探讨20S蛋白酶体β5亚单位（PSMB5）对体外扩增培养人骨髓间充质干细胞（humanbone marrow mesenchymal stem cells, hBMSCs）增殖能力的影响，明确调控hBMSCs细胞活力的有效靶点，从而为干细胞临床移植奠定实验基础。 方法：体外分离培养hBMSCs，依据体外传代次数将其分为早期组（1-3代）和晚期组（12-14代）。1.检测PSMB5在早期和晚期hBMSCs的表达变化。（1）通过溴脱氧尿嘧啶核苷（BrdU）掺入实验和流式细胞术检测早期和晚期hBMSCs增殖能力。（2）应用荧光酶标仪检测早期和晚期hBMSCs20S蛋白酶体活性。（3）应用real-time PCR技术分析蛋白酶体β亚单位PSMB1、 PSMB5和PSMB6的表达变化；应用Western blotting检测早期和晚期20S蛋白酶体和PSMB5亚单位的表达变化。2.构建携带绿色荧光蛋白（GFP）基因的PSMB5-shRNA慢病毒载体，并设错义序列对照，感染早期hBMSCs，观察PSMB5基因沉默对hBMSCs增殖能力的影响。（1）慢病毒感染细胞后倒置荧光显微镜观察绿色荧光蛋白表达情况，判断感染效率。（2）应用real-time PCR和Western blotting分别从基因和蛋白水平检测PSMB5抑制效率。（3）荧光酶标仪检测20S蛋白酶体活性的变化。（4）应用Ki67和BrdU免疫荧光染色检测hBMSCs增殖率，Western blotting检测细胞周期相关蛋白（Cyclin D1）的表达变化。 结果：1.（1）BrdU掺入实验结果证明早期hBMSCs BrdU阳性率为68.92±7.08%，晚期hBMSCs阳性率为27.67±2.76%，提示传代晚期hBMSCs增殖能力较早期细胞显著降低(P0.01)；流式细胞仪分析结果显示，晚期hBMSCs S期细胞(4.42±0.98%)较早期hBMSCs(13.72±1.58%)显著减少(P0.01)，细胞增殖指数较早期细胞降低11.02±1.88%(P0.05)，进一步说明扩增培养晚期hBMSCs增殖活力降低。（2）晚期hBMSCs20S蛋白酶体活性较早期显著降低52.99±13.08%(P0.01)，提示20S蛋白酶体活性可能与hBMSCs细胞活力有关。（3）real-time PCR结果表明，传代晚期细胞蛋白酶体亚基PSMB1、PSMB5表达水平较早期分别降低13.0±1.0%和37.0±8.0%(P0.01)，且PSMB5下降最为明显；同时，Western blotting实验进一步表明晚期hBMSCs20S蛋白酶体和PSMB5表达水平分别比早期下降54.51±5.94%(P0.05)和52.99±13.08%(P0.05)，提示20S蛋白酶体核心亚单位PSMB5可能是调控hBMSCs细胞活力的关键因素。2.（1）PSMB5-shRNA慢病毒感染早期hBMSCs，24h后隐约可见部分细胞发出绿色荧光，但是信号较弱；72h后荧光信号增强，，约90%以上细胞可见绿色荧光。（2）real-time PCR和Western blotting结果证实，PSMB5-shRNA组PSMB5mRNA和蛋白的表达水平分别较GFP对照组下调93.3±0.34%(P0.01)和56.9±13.31%(P0.05)，提示慢病毒介导的shRNA能够有效下调目的基因PSMB5的表达水平。（3）PSMB5-shRNA组20S蛋白酶体活性较GFP对照组降低69.87±1.32%(P0.05)，提示下调PSMB5表达水平能够降低早期hBMSCs20S蛋白酶体活性。（4）Ki67免疫荧光染色结果显示，PSMB5-shRNA组Ki67阳性率较GFP对照组降低76.18±12.37%(P0.01)；同时BrdU掺入实验结果也证明，PSMB5-shRNA组BrdU阳性率26.14±8.13%较GFP对照组49.53±11.18%显著降低(P0.01)，表明PSMB5基因沉默能够抑制早期hBMSCs增殖能力。此外，Western blotting实验结果证明PSMB5-shRNA组细胞周期相关蛋白Cyclin D1表达水平较GFP对照组降低54.55±5.49%(P0.05)，提示PSMB5基因下调可能通过抑制细胞周期进程，从而影响hBMSCs增殖能力。 结论：（1）晚期hBMSCs增殖能力降低，并且20S蛋白酶体活性及其核心亚单位PSMB5表达水平下降。（2）应用基因沉默技术手段下调PSMB5表达水平，能够抑制早期hBMSCs增殖能力，提示PSMB5可能是影响hBMSCs增殖活力的关键靶点。
[Abstract]:Objective: To investigate the effect of 20S proteasome beta 5 subunit (PSMB5) on the proliferation of human bone marrow mesenchymal stem cells (humanbone marrow mesenchymal stem cells, hBMSCs) in vitro, and to clarify the effective targets for regulating the viability of hBMSCs cells, so as to lay an experimental basis for the clinical transplantation of stem cells.
Methods: hBMSCs was isolated and cultured in vitro. According to the number of passages in vitro, it was divided into early group (1-3 generation) and late group (12-14 generation) to detect the expression changes of PSMB5 in early and late hBMSCs. (1) detection of early and late hBMSCs proliferation by bromodeoxyuridine (BrdU) incorporation and flow cytometry. (2) the use of fluorescence enzyme labeling method. The activity of early and late hBMSCs20S proteasome was measured. (3) real-time PCR technique was used to analyze the expression of proteasome beta subunit PSMB1, PSMB5 and PSMB6, and Western blotting was used to detect the expression changes of early and late 20S proteasome and PSMB5 subunits and to construct the lentivirus carrying green color fluorescent protein (GFP) gene. Vector, and missense sequence control, infect early hBMSCs, observe the effect of PSMB5 gene silencing on hBMSCs proliferation ability. (1) the expression of green fluorescent protein was observed by inverted fluorescence microscope after lentivirus infection, and infection efficiency was judged. (2) real-time PCR and Western blotting were used to detect PSMB5 inhibition from gene and protein levels, respectively. Efficiency. (3) the changes in the activity of 20S proteasome were detected by the fluorescent enzyme labeling apparatus. (4) the proliferation rate of hBMSCs was detected by Ki67 and BrdU immunofluorescence staining, and the expression of cell cycle related protein (Cyclin D1) was detected by Western blotting.
Results: the results of 1. (1) BrdU incorporation showed that the positive rate of early hBMSCs BrdU was 68.92 + 7.08% and the positive rate of late hBMSCs was 27.67 + 2.76%, suggesting that the proliferation ability of late hBMSCs was significantly lower than that of early cells (P0.01). The results of flow cytometry showed that the late hBMSCs S phase cells (4.42 + 0.98%) were significantly higher than those of early hBMSCs (13.72 + 1.58%). Decrease (P0.01), the cell proliferation index decreased by 11.02 + 1.88% (P0.05), further indicating that the proliferation activity of advanced hBMSCs was reduced. (2) late hBMSCs20S proteasome activity was significantly reduced by 52.99 + 13.08% (P0.01), suggesting that the viability of 20S proteasome may be related to the viability of hBMSCs cells. (3) real-time PCR results showed that The advanced cell proteasome subunit PSMB1, PSMB5 expression level decreased by 13 + 1% and 37 + 8% (P0.01), respectively, and the PSMB5 decreased most obviously. Meanwhile, Western blotting experiment further indicated that the expression level of late hBMSCs20S proteasome and PSMB5 decreased by 54.51 + 5.94% (P0.05) and 52.99 + 13.08% (P0.05), respectively. The core subunit PSMB5 of 20S proteasome may be a key factor in the regulation of the activity of hBMSCs cells,.2. (1) PSMB5-shRNA lentivirus infection early hBMSCs. After 24h, some cells emit green fluorescence, but the signal is weak; after 72h, the fluorescence signal is enhanced, and more than 90% cells can be seen green fluorescence. (2) real-time PCR and Western blotting The results showed that the expression level of PSMB5mRNA and protein in PSMB5-shRNA group was down 93.3 + 0.34% (P0.01) and 56.9 + 13.31% (P0.05), respectively, suggesting that lentivirus mediated shRNA could effectively reduce the expression level of PSMB5 in the target gene. (3) the activity of 20S proteasome in PSMB5-shRNA group decreased by 69.87 + 1.32% (P0.05) in the GFP control group, suggesting down regulation. The expression level of PSMB5 could reduce the activity of early hBMSCs20S proteasome. (4) the results of Ki67 immunofluorescence staining showed that the positive rate of Ki67 in the PSMB5-shRNA group was 76.18 + 12.37% lower than that of the GFP control group (P0.01), and the BrdU incorporation test results also showed that the positive rate of BrdU in the PSMB5-shRNA group was 26.14 + 8.13% compared with the GFP control group (49.53 + 11.18%) (P0.01). It was indicated that PSMB5 gene silencing could inhibit the early hBMSCs proliferation ability. In addition, the Western blotting test results showed that the expression level of cell cycle related protein Cyclin D1 in PSMB5-shRNA group was 54.55 + 5.49% (P0.05) lower than that of the GFP control group, suggesting that the PSMB5 gene downregulation may affect the hBMSCs proliferation by inhibiting the cell cycle process.
Conclusion: (1) the proliferation ability of late hBMSCs decreased, and the activity of 20S proteasome and the expression level of PSMB5 in the core subunit decreased. (2) the application of gene silencing technique to reduce the expression of PSMB5 could inhibit the early hBMSCs proliferation ability, suggesting that PSMB5 may be the key target for the proliferation of hBMSCs.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R329.2

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