【作者】 朱海燕；

【导师】 舒建昌；

【作者基本信息】 暨南大学 ， 内科学， 2010， 硕士

【摘要】 目的1.观察NGF对HSC增殖及凋亡的影响,并探讨其可能的作用机制。2.观察NGF作用后HSC中NGF、p75NTR及凋亡相关基因P53、Bcl-2、Caspase-3蛋白表达的情况。3.观察NGF对HSC合成I、III型胶原的影响。方法HSC与不同浓度的NGF孵育后,XTT比色法检测NGF对HSC增殖的影响,流式细胞术分析NGF对HSC细胞周期的影响；流式细胞术、吖啶橙染色、透射电镜检测细胞凋亡；免疫荧光法检测NGF、p75NTR表达的情况；细胞免疫化学法检测HSC中凋亡相关基因P53、Bcl-2、Caspase-3蛋白表达的情况；ELISA法检测HSC培养上清中I、III型胶原的含量。结果(100、200、400)ng/mL浓度时NGF对HSC的抑制作用经XTT法测得OD值分别为0.66±0.03、0.69±0.03和0.66±0.03,与对照组(0.73±0.01)相比下降具有统计学意义(P<0.05),但无浓度依赖性(P>0.05)。(100、200、400)ng/mLNGF作用于HSC24h后,G2期比例分别为(14.83±5.41)%、(14.73±2.50)%和(14.87±2.06)%,与对照组[(7.47±4.39)%]相比明显增加(P<0.05)。流式细胞术检测100ng/mLNGF作用HSC24h后凋亡率为(22.36±9.51)%,与对照组[(5.88±1.36)%]比较明显增高,差异有统计学意义(P<0.05)。吖啶橙染色100ng/mLNGF作用HSC后细胞数量减少,且细胞体积缩小、形态不规整,细胞核固缩,荧光增强,可见浓缩致密的暗绿色或黄绿色荧光。透射电镜可以见到100ng/mLNGF作用HSC后,凋亡细胞体积较正常细胞明显缩小,核染色质浓缩,沿核膜排列,细胞器也发生浓缩,并可以见到凋亡小体形成。100ng/mLNGF作用HSC后免疫荧光显示NGF表达增多,其平均光密度值(6.53+1.40)与对照组(1.77+0.17)比较差异有统计学意义(P<0.05),而p75NTR表达无明显变化,其平均光密度值(4.24±0.38)与对照组(3.52±0.36)比较差异无统计学意义(P>0.05)。凋亡相关蛋白P53、Caspase-3的阳性细胞百分率分别为(78.41±4.00)%、(39.26+1.57)%,与对照组(34.96±3.84)%、(9.27±1.01)%比较明显增高,而Bcl-2表达实验组阳性细胞百分率为(18.12±1.38)%,较对照组[(91.53±2.98)%]降低,差异有统计学意义(P<0.05)。(100、200、400)ng/mLNGF作用HSC24h后培养上清中I型胶原的含量分别为(3.27±0.41)ng/mL、(3.45±0.17)ng/mL和(3.88±0.71)ng/mL,与对照组[(6.28±0.81)ng/mL]相比明显减少(P<0.05), III型胶原的含量分别为(3.05±0.52)ng/mL、(3.06±0.41)ng/mL和(2.98+0.48)ng/mL,与对照组[(3.89±0.29)ng/mL]相比含量下降具有统计学意义(P<0.05)。结论1.NGF可以抑制HSC增殖,诱导其发生凋亡。2.NGF可能通过使HSC细胞周期停滞于G2期,而发挥抑制HSC增殖的作用。3.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。4.NGF可能通过使凋亡相关基因P53、Caspase-3表达上调、Bcl-2表达下调,从而诱导HSC凋亡5.NGF可以抑制HSC合成I、III型胶原。更多还原

【Abstract】 Objective1. To investigate the effect of NGF on proliferation and apoptosis of HSC in vitro and explore the possible mechanism.2. To study the expressions of NGF and p75NTR proteins and apoptosis-regulating proteins Caspase-3, P53 and Bcl-2 of HSC after treated with NGF.3. To investigate the effect of NGF on the synthesis of collagen I and III by HSC.MethodsHSC was incubated with different concentrations of NGF. The effect of NGF on cell proliferation was observed by XTT colormetric assay. HSC cell cycle was analyzed by flow cytometry (FCM). HSC apoptosis was identified by FCM, acridine orange staining and transmission electron microscopy (TEM). Expressions of NGF and p75NTR were detected by immunofluorescence. The expressions of apoptosis-regulating proteins Caspase-3, P53 and Bcl-2 of HSC after apoptosis induced by NGF were examined by immunohistochemical staining. Collagen I and III in the supernate of medium secreted by HSC were detected by enzyme-linked immunosorbent assay.Results(100,200,400)ng/mL NGF incubated with HSC for 24h could inhibit proliferation of HSC(P<0.05) without concentration-dependent(P>0.05):XTT colormetric assay showed that optical density of NGF groups were 0.66±0.03, 0.69±0.03 and 0.66±0.03, respectively, but in control group it was 0.73±0.01. After treating HSC with NGF at the concentration of 100,200,400ng/mL, the number of HSC in G2 phase were (14.83±5.41)%, (14.73±2.50)% and (14.87±2.06)%, respectively. They were significantly higher than control group in which the number of HSC in G2 phase was (7.47±4.39)% (P<0.05). Apoptosis index of HSC was higher than that of control group [(22.36±9.51)% vs (5.88±1.36)%] afer theated with NGF (100ng/mL) detected by flow cytometry (P<0.05). Acridine orange staining showed fluorescence enhancement, dense concentration of dark green or yellowish green fluorescence, the shape of HSC became shrinkage and irregular in NGF group. Cell shrinkage, chromatin condensation and (or) ranked along inside of nuclear membrane and apoptosis body could be found by transmission electron microscope. When HSC was stimulated with 100ng/mL NGF for 24h, immunofluorescence assay showed that the average optical density of NGF increased significantly compared with the 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). After incubating with 100ng/mL NGF for 24h, the positive expression rates of P53 and Caspase-3 of HSC increased significantly compared with those of control group:(78.41±4.00)% vs (34.96±3.84)%, (39.26±1.57)% vs (9.27±1.01)%, respectively (P<0.05). The positive expression rate of Bcl-2 protein of HSC decreased significantly compared with that of control group [(18.12±1.38)% vs (91.53±2.98)%] (P<0.05). When HSC was incubated with (100,200,400)ng/mL NGF for 24h, the contents of collagen I in the supernate of medium were (3.27±0.41)ng/mL, (3.45±0.17) ng/mL and (3.88±0.71) ng/mL, respectively, which were less than that of the control group [(6.28±0.81)ng/mL](P<0.05); as well as collagen I in NGF groups the contents of collagen III were (3.05±0.52) ng/mL, (3.06±0.41) ng/mL and (2.98±0.48) ng/mL, respectively, which were also significantly less than that of the control group [(3.89±0.29) ng/mL] (P<0.05).Conclusions1. NGF could significantly inhibit the proliferation and induce the apoptosis of HSC.2. NGF could make HSC cell cycle arrest in G2 phase, it maybe the mechanism of NGF inhibited the proliferation of HSC.3. NGF could be used as an initiating factor and effect factor to increase the expression of NGF on HSC, but no significant effect on p75NTR expression.4. The mechanism of NGF to induce HSC apoptosis may be associted with the up-regulating expression of Caspase-3, P53 and down-regulating expression of Bcl-2 on HSC. 5. NGF could inhibit HSC from synthesizing collagenⅠand collagenⅢ.更多还原