hTERT基因修饰对人外周血树突状细胞生物学行为的影响

The biologic behavior changes in dendritic cells after modification by hTERT gene

目的观察人端粒酶逆转录酶(hTERT)基因修饰对树突状细胞生物学行为的影响。方法用负载hTERT目的基因的复制缺陷型腺病毒(rAd-hTERT)感染原代培养的人树突状细胞(DC)以获取hTERT基因修饰的DC(DC-hTERT),采用免疫组化及Westernblot法检测DC-hTERT内hTERT及增殖细胞核抗原(PCNA)的表达;采用流式细胞术检测DC-hTERT表面成熟标志的变化;采用MTT法检测DC-hTERT的增殖能力。结果rAd-hTERT感染DC后hTERT蛋白表达明显增加;流式细胞术检测显示,感染Ad-hTERT后DC表面成熟指标CD83及CD86无明显变化;成熟DC经hTERT修饰后的生长曲线显示,DC-hTERT在前2周数量稍有增加,但DC/PBS及DC/rAd-LacZ数量明显下降,在第3周三者数量均有下降趋势,但DC-hTERT组细胞数量仍明显多于其他两组;Westernblot检测显示DC-hTERT内PCNA的表达明显增强。结论hTERT基因表达上调后,DC增殖能力明显增强,衰老速度延缓。

Objective To investigate the changes in biologic behavior of dendritic cells （DCs） after being modified by human telomerase reverse transcriptase （hTERT） gene. Methods In order to obtain the hTERT gene modified DCs, DCs were transfected with a replicationdeficient recombinant adenovirus expression vector of hTERT. Then the expression to hTERT and PCNA was assessed by by Western blot, mature markers on DCs surface were detected by flow cytometry, and the proliferative capacity was determined by MTT method. Results Immunohistochemical staining and Western blotting showed that the expression of hTERT was upregulated obviously in DCs after being modified by hTERT gene. Flow cytometry indicated that the expression of CD83 and CD86 remained unchanged. The DC growth curve showed that the number of DC-hTERT was increased slightly in the first 2 weeks, meanwhile the number of DC/rAd-LacZ and DC was decreased obviously. Although the number in 3 groups was all decreased in the third week, the number of DC/rAd-hTERT was still greater than the other control groups. It was also found that the expression of PCNA was increased in DC/rAd-hTERT compared with that of immature DC, mature DC or DC/rAd-LacZ by Western blot. Conclusion After rAd-hTERT modification, life span of DC in vitro is extended and proliferative capacity is enhanced.