神经生长因子诱导肝星状细胞凋亡及其相关机制的研究

Study on the mechanism of apoptosis of hepatic stellate cells induced by nerve growth factor

目的观察神经生长因子（nerve growthfactor，NGF）对大鼠肝星状细胞（hepatic stellatecell，HSC）凋亡的影响，并探讨其可能的作用机制。方法体外培养大鼠肝星状细胞株HSC—T6，将HSC—T6与100ng／mlNGF孵育24h后，流式细胞术检测细胞凋亡；细胞免疫化学法检测HSC中凋亡相关基因P^53、Bcl-2、Caspase-3蛋白表达的情况；免疫荧光法检测NGF、神经生长因子低亲和力受体p75NTR表达的情况。结果NGF作用HSC后凋亡率较对照组明显增高[（22．364±9．51）％VS（5．88±1．36）％，P〈0．05]。凋亡相关蛋白P^53、Caspase-3的阳性细胞百分率与对照组比较明显增高[（78．41±4．00）％、（39．26±1．57）％VS（34．96±3．84）％、（9．27±1．01）％，P〈0．05]，而Bcl-2表达实验组阳性细胞百分率较对照组降低[（18．12±1．38）％vs（91．53±2．98）％，P〈0．05]。NGF作用HSC后NGF表达增多（6．53±1．40vs1．77±0．17，P〈0．05），而p75NTR表达无明显变化（3．52±0．36VS4．24±0．38，P〉0．05）。结论NGF可能通过使凋亡相关基因p、Caspase-3表达上调、Bcl-2表达下调，而诱导HSC凋亡。NGF作用HSC后可作为始动因子和效应因子增加NGF的表达，而对p75NTR表达无影响。

Objective To investigate the effect of NGF on apoptosis of HSC in vitro and explore the possible mechanism. Methods HSC was incubated with different concentrations of NGF. HSC apoptosis was identified by FCM. The expressions of apoptosis-regulating proteins Caspase-3, p53 and Bcl-2 of HSC after apoptosis induced by NGF were examined by immunohistochemieal staining. Expressions of NGF and p75NTR were detected by immunofluorescence. Results Apoptosis index of HSC was higher than that of control group [（22.36 ±9.51）% vs （5.88 ± 1.36）%] after treated with NGF （100 ng/ml） （ P 〈0. 05 ）. After incubating with 100 ng/ml NGF for 24 h, the positive expression rates of pS3 and Caspase-3 of HSC increased significantly than those of control group [ （78. gl ＋ 4. 00） % vs （ 34. 96 ± 3.84） %, （39. 26 ＋ 1.57）% vs （9. 27 ± 1.01）%, P 〈0. 05]. The positive expression rate of Bcl-2 protein of HSC signifi- cantly decreased compared with that of control group （ 18. 12 ± 1.38 ） % vs （91.53 ± 2. 98 ） % （ P 〈 O. 05）. When HSC was stimulated with 100 ng/ml NGF for 24 h, the average optical density of NGF in-creased significantly than control group （6. 53＋ 1.40 vs 1.77 ±0. 17） （ P 〈0. 05 ）, while the expression of p75NTR was not significantly; changed （3.52 ＋0. 36 vs 4. 24 ±0. 38） （ P 〉0. 05）. Conclusions The mechanism of NGF to induce HSC apoptosis may be associated with the up-regulating expression of Caspase- 3, p53 and down-regulating expression of Bcl-2 on HSC. NGF could be used as an initiating factor and effect factor to increase the expression of NGF on HSC, but it had no significant effect on o75NTR exnression.