海珠抗纤方治疗慢性乙型肝炎肝纤维化的临床研究和机制探讨
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摘要
目的:
观察海珠抗纤方治疗慢乙肝肝纤维化的疗效，检测该方治疗前后外周血T细胞亚群、CD4~+CD25~+Foxp3~+调节性T淋巴细胞、TGF-β1水平、HBV特异性细胞毒性T淋巴细胞的水平，探讨该方治疗慢性乙型肝炎肝纤维化的免疫机制，为中药从免疫的角度逆转肝纤维化提供理论基础，也为中医微观辩证提供依据。
方法：
1、将慢乙肝肝纤维化患者122例随机分为三组。治疗组62例，给予海珠抗纤方治疗，每日1剂，每天3次口服；对照1组60例，给予安络化纤丸治疗，6g/次，每日3次；对照2组给予还原型谷胱甘肽片治疗，每次0.4g口服，每天3次，三组疗程均为24周。观察各组治疗前后临床症状体征变化，检测各组治疗前后肝功能、血清HBVDNA水平、血清肝纤四项、肝脏Fibroscan弹性值的的变化。
2、将60例慢乙肝肝纤维化患者随机分为二组，治疗组30例，给予海珠抗纤方治疗，每日1剂，每天3次口服；对照组30例，给予还原型谷胱甘肽片治疗，每次0.4g口服，每天3次，两组疗程均为24周；选取健康体检成人30人为健康对照组。入选60例慢性乙型肝炎患者中有40例次行肝穿刺术，做肝组织病理学检查。检测各组治疗前后肝纤四项的水平；分别以细胞内表达IFN-γ作为Th1细胞亚群的标志，细胞内表达IL-4作为Th2细胞亚群的标志，通过流式细胞仪细胞内细胞因子染色法检测各组治疗前后Th1/Th2细胞亚群的变化，通过ELISA双抗夹心法检测各组治疗前后血清IFN-γ、IL-4水平变化；分析Th1/Th2细胞亚群与肝纤四项、肝组织病理纤维化分期之间的关系；观察海珠抗纤方对Th1/Th2细胞亚群的影响。
3、在第二部分研究的基础之上，通过流式细胞仪细胞内细胞因子染色法检测实验二中治疗组和对照组治疗前后血清CD4~+CD25~+Foxp3~+Treg细胞水平，通过ELISA双抗体夹心法检测治疗前后二组血清TGF-β1水平，进一步研究血清CD4~+CD25~+Foxp3~+Treg、TGF-β1水平与肝组织病理学及肝纤四项之间的关系，观察海珠抗纤方对慢乙肝肝纤维化CD4~+CD25~+Foxp3~+Treg、TGF-β1的影响。
4、应用HLA-A2作为过筛指标，经HLA抗体检测技术筛选HLA-A2（+）慢乙肝患者共62例。入选HLA-A2阳性慢乙肝患者随机分为二组。治疗组32例，给予海珠抗纤方治疗，每日1剂，每天3次口服；对照组30例，给予还原型谷胱甘肽片治疗，每次0.4g口服，每天3次，疗程均为24周。入选患者中有30例行肝穿术，做肝组织病理检查，检测治疗前后血清HBVDNA定量及肝纤四项水平；通过流式细胞仪免疫荧光法检测二组治疗前后CD4~+T淋巴细胞亚群、CD8+T淋巴细胞亚群、CD4~+/CD8+比值；通过流式细胞仪MHC-肽-五聚体法检测二组治疗前后HBV特异性细胞毒性T淋巴细胞水平及分泌IFN-γ功能的变化；分析T淋巴细胞亚群、HBV特异性细胞毒性T淋巴细胞与肝纤四项、肝组织纤维化分期之间的关系，观察海珠抗纤方对T淋巴细胞亚群和HBV特异性细胞毒性T淋巴细胞的影响。
结果：
1、海珠抗纤方治疗后慢乙肝肝纤维化患者临床症状及体征较治疗前明显好转(P＜0.05)，肝功能指标（ALT、AST、A/G、TBIL）较治疗前显著好转(P＜0.05)，其中治疗后ALT、AST改善上优于安络化纤丸组（对照1组），与西药护肝组（对照2组）比较无显著性差异(P＞0.05)；治疗后A/G比值改善上优于安络化纤丸组和西药护肝组（对照2组）(P＜0.05)。海珠抗纤方治疗后血清HBVDNA定量水平较治疗前显著下降(P＜0.05)；安络化纤丸组及西药护肝组治疗前后血清HBVDNA定量无明显改变(P＞0.05)。海珠抗纤方组及安络化纤丸组治疗后血清肝纤四项指标均较治疗前显著下降（P＜0.05），肝脏Fibroscan检测弹性值均较治疗前明显下降（P＜0.05），两组治疗后血清肝纤四项及Fibroscan弹性值比较无显著性差异(P＞0.05)；西药护肝组（对照2组）治疗前后肝纤四项及Fibroscan检测弹性值虽略有下降，但经统计学比较无显著性差异(P＞0.05)。
2、慢乙肝肝纤维化患者Th1、Th2细胞亚群水平及Th1/Th2比值均较健康对照组显著降低（P＜0.05），与健康对照组相比Th1下降更为明显，表现为Th2细胞亚群水平相对占优势。Th1细胞亚群水平与肝纤四项成负相关（r=－0.61，P＜0.05），并且随着肝脏纤维化分期的增加而下降；Th2细胞亚群水平与肝纤四项成正相关（r=0.43，P＜0.05），并且随着肝脏纤维化分期的增加而上升；Th1/Th2比值分别与肝纤四项、肝纤维化分期成负相关，相关系数分别为r=－0.52，P＜0.05，r=－0.68，P＜0.05。随着肝纤维化的进展，机体Th1细胞亚群水平逐渐降低，Th2细胞亚群水平逐渐升高，Th1/Th2比值逐渐降低，机体免疫应答出现由Th1向Th2漂移的现象。海珠抗纤方治疗后Th1细胞亚群水平及Th1/Th2比值均明显升高（P＜0.05），而对照组治疗前后Th1、Th2细胞亚群水平及Th1/Th2比值均无显著性改变（P＞0.05）。
3、慢乙肝肝纤维化患者治疗前血清肝纤四项、TGF-β1及CD4~+CD25~+Foxp3~+Treg细胞亚群水平较健康对照组明显升高（P＜0.05），治疗组治疗后血清肝纤四项、TGF-β1及CD4~+CD25~+Foxp3~+Treg细胞亚群水平较治疗前显著降低（P＜0.05）；对照组治疗前后血清肝纤四项、TGF-β1及CD4~+CD25~+Foxp3~+Treg细胞亚群水平无明显改变（P＞0.05）。血清TGF-β1水平与肝纤四项成正相关，（相关系数分别为:rHA=0.82, P＜0.05；rLN=0.69, P＜0.05；rPCⅢ=0.72,P＜0.05；rC-Ⅳ=0.65, P＜0.05）；血清TGF-β1水平与肝脏病理分期成正相关（r=0.84，P＜0.05），血清CD4~+CD25~+Foxp3~+Treg细胞亚群水平与TGF-β1水平及肝脏病理分期成显著正相关（相关系数为r=0.58，P＜0.05；r=0.55，P＜0.05）。
4、慢乙肝肝纤维化患者治疗前CD4~+T比例及CD4~+/CD8+比值较健康对照组显著下降(P＜0.05)，CD8+T比例明显升高（P＜0.05)，血清CD4~+T淋巴细胞比例及CD4~+/CD8+比值与肝脏纤维化病理分期成负相关（相关系数分别为rCD4~+=－0.53，P＜0.05；rCD4~+/CD8+=－0.59，P＜0.05）；血清CD8+T淋巴细胞比例与肝脏纤维化病理分期成正相关（rCD8+=0.42，P＜0.05）。随着肝纤维化程度的加重，CD4~+T淋巴细胞比例及CD4~+/CD8+比值逐渐下降，CD8+T淋巴细胞比例逐渐上升，T细胞亚群的紊乱逐渐加重。海珠抗纤方治疗后血清CD4~+T淋巴细胞比例、CD4~+/CD8+比值、HBVcore18-27特异性细胞毒性T淋巴细胞频数及产生IFN-γ的频数较治疗前显著提高（P＜0.05）；CD8+T淋巴细胞比例、血清HBVDNA水平较治疗前显著下降（P＜0.05）；对照组治疗治疗前后T淋巴细胞亚群、HBVDNA水平、HBVcore18-27特异性细胞毒性T淋巴细胞频数及产生IFN-γ的频数均无显著性差异（P＞0.05）。
结论：
1、海珠抗纤方能够改善慢性乙型肝炎肝纤维化患者的临床症状和体征，改善肝功能，升高A/G比值，降低血清HBVDNA含量，降低血清肝纤四项和改善肝脏硬度，其抗纤维化的作用与安络化纤丸相比无显著性差异，但在抑制病毒复制和改善肝功能指标上优于安络化纤丸。
2、海珠抗纤方可以升高慢乙肝肝纤维化患者血清Th1细胞亚群水平及Th1/Th2比值，从而纠正Th1/Th2失衡，使机体免疫功能向Th1细胞亚群占优势的方向逆转。还原性谷胱甘肽片对纠正Th1/Th2失衡无明显作用。
3、海珠抗纤方能显著降低慢乙肝肝纤维化患者血清CD4~+CD25~+Foxp3~+Treg及TGF-β1水平，对血清CD4~+CD25~+Foxp3~+Treg及TGF-β1水平有一定调节作用，可以阻断CD4~+CD25~+Foxp3~+Treg-TGF-β1-HSC三者之间的循环反应，逆转肝纤维化患者HSC的免疫微环境，促进肝纤维化的恢复。
4、海珠抗纤方通过提高CD4~+T淋巴细胞比例、CD4~+/CD8+比值，降低CD8+T淋巴细胞比例，从而减轻肝细胞损伤，促进慢性乙型肝炎肝纤维化的恢复。同时可以提高慢乙肝肝纤维化患者HBV抗原特异性CTL的数量及分泌IFN-γ的功能。海珠抗纤方通过纠正慢乙肝肝纤维化患者的免疫失衡既有利于控制HBV病毒复制，也有利于逆转乙型肝炎肝纤维化。
Objective:Observe the clinical effect of HZKXF on liver fibrosisin patients of chronic hepatitis B.Detect the serum level of Th1/Th2,CD4~+CD25~+Foxp3~+Treg, TGF-β1,T lymphocyte subsets and HBV specificCTL.Probe into the immunologic mechanism of HZKXF on fibrosis.
Methods:
1、122patients of chronic hepatitis B were divided into3groupsrandomly.The treatment group was treated by HZKXF(everyday1agent,3times a day).The control group1was treated by Anluohuaxianpills (6grams evevy time,3times a day). Control group2wastreated by reduced glutathione tablets(0.4grams evevy time,3times a day).The courses of treatment were all24weeks in everygroup.Detect the level of liver function,HBVDNA quantity andHA,LN,PCⅢ,C-Ⅳ,liver elasticity number before and after thetreatment.Observe the clinical effect of HZKXF on liver fibrosis.
2、60patients of chronic hepatitis B were divided into2groupswith random.The treatment group was treated by HZKXF(everyday1agent,3times a day).The control group was treated by reducedglutathione tablets(0.4grams evevy time,3times a day). Thecourses of treatment were both24weeks.30healthy people were thehealthy control group.There are40times liver puncture among60patients. Detect the level of HA,LN,PCⅢ,C-Ⅳ, the serum level ofIFN-,IL-4,Th1/Th2before and after the treatment.Analyse the correlativity relations between Th1/Th2, serum hepatic fibrosisindexes and the stage of liver fibrosis. Observe the effect of HZKXFon Th1/Th2.
3、Based on the second experiment,using the flow cytometryintracellular cytokine staining method to dectect the level ofCD4~+CD25~+Foxp3~+Treg in serum before and after the treatment.Usingthe ELISA double antibody sandwich method to dectect the level ofTGF-β1in serum before and after the treatment. Analyse the corr-elativity relations between CD4~+CD25~+Foxp3~+Treg,TGF-β1, serumhepatic fibrosis indexes and the stage of liver fibrosis. Observethe effect of HZKXF on CD4~+CD25~+Foxp3~+Treg and TGF-β1.
4、Application of HLA-A2as a screening index，62patients ofHLA-A2antibody(+) were divided into2groups randomly in patientswith chronic hepatitis B. There were32patients in the treatmentgroup.The treatment group was treated by HZKXF (everyday1agent,3times a day). There were30patients in the control group.Thecontrol group was treated by reduced glutathione tablets(0.4gramsevevy time,3times a day).The courses of treatment were all24weeksin every group.Using flow cytometry to dectect the level of CD4~+,CD8+and CD4~+/CD8+in serum before and after treatment.Using MHC-HBVcore18-27-pentmer to dectect the level of HBV specific CTL and the itsfunction of secreting IFN-γin serum before and after the treatment.Analyse the correlativity relations between T lymphocyte subsets,HBV specific CTL,HBVDNA quantity serum hepatic fibrosis indexes andthe stage of liver fibrosis.
Results:
1、The level of liver function was improved obviously after HXKXF treatment(P＜0.05). The level of ALT and AST were improvedmore obviously in the treatment group than in the control group1.There were no significant differences on the level of ALT and ASTbetween the treatment group and the control group2after thetreatment(P＞0.05).The level of A/G was improved more obviously inthe treatment group than in the control group1and control group.
2、The level of HBVDNA quantity was decreased more obviouslyin the treatment group than in the countrol group1and countrolgroup2. There were no significant differences on the level ofHBVDNA quantity both in the control group1and control group2afterthe treatment(P＞0.05). The level of liver fibrosis indexes inserum and liver elasticity numbers detecting with Fibroscan weredecreased obviously after HXKXF and Anluohuaxian pills treatment(P＜0.05).There were no significant differences between the2groupson the level of liver fibrosis indexes in serum and liver elasticitynumbers(P＞0.05).There were no significant differences on thelevel of liver fibrosis indexes in serum and liver elasticitynumbers after the treatment in the control group2(P＞0.05).
3、The level of Th1、Th2and Th1/Th2decreased obviously comparedwith the healthy control group(P＜0.05).The level of Th1decreasedmore obviously than the level of Th2. The level of Th1negativelycorrelated with the liver fibrosis indexes in serum（r=－0.71，P＜0.05）,it declined with the stage of liver fibrosis rised.Th2positively correlated with the liver fibrosis indexes in serum（r=0.58，P＜0.05）,it elevated the stage of liver fibrosis rised.Th1/Th2negatively correlated with the liver fibrosis indexes inserum（r=－0.82，P＜0.05） and the stage of liver fibrosis(r=－0.84，P＜0.05).The level of Th1and Th1/Th2elevated after HZKXF treatment（P＜0.05）.The level of Th2decreased after HZKXFtreatment（P＜0.05）. There were no significant differences on thelevel of Th1, Th2and Th1/Th2after the treatment in the controlgroup (P＞0.05).
4、 The level of the liver fibrosis indexes,TGF-β1andCD4~+CD25~+Foxp3~+Treg in serum rised obviously compared with thehealthy control group(P＜0.05). The level of the liver fibrosisindexes,TGF-β1and CD4~+CD25~+Foxp3~+Treg in serum decreased afterHZKXF treatment(P＜0.05). There were no significant differences onthe level of the liver fibrosis indexes,TGF-β1andCD4~+CD25~+Foxp3~+Treg in serum after the treatment in the control group(P＞0.05).The level of TGF-β1positively correlated with the liverfibrosis indexes in serum（rHA=0.56, P＜0.05；rLN=0.58, P＜0.05；rPC Ⅲ=0.56,P＜0.05；rC-Ⅳ=0.48, P＜0.05）. The level of TGF-β1positively correlated with the stage of liver fibrosis（r=0.51，P＜0.05）.The level of CD4~+CD25~+Foxp3~+Treg positively correlatedwith the level of TGF-β1in serum.
5、The level of CD4~+T and CD4~+/CD8+in serum decreased,the levelof CD8+rised obviously compared with the healthy control groupbefore the treatment(P＜0.05).The level of CD4~+T and CD4~+/CD8+negatively correlated with the stage of liver fibrosis(rCD4~+=－0.53，P＜0.05；rCD4~+/CD8+=－0.59，P＜0.05）. The level of CD8+positivelycorrelated with the stage of liver fibrosis（rCD8+=0.55，P＜0.05）.Thelevel of CD4~+, CD4~+/CD8+, HBVcore18-27specific CTL and seretingIFN-γ improved after HXKXF treatment（P＜0.05）.The level of CD8+and HBVDNA quantity decreased after HXKXF treatment（P＜0.05）.There were no significant differences on the level of T lymphocyte subsets, HBVcore18-27specific CTL and sereting IFN-γin serumafter the treatment in the control group (P＞0.05).
Conclusion:
1、HZKXF can improved the liver function,decrease the levelof HBVDNA quantity,liver fibrosis indexes, liver elasticitynumbers. There were no significant differences on anti-hepaticfibrosis effects(P＞0.05).Itis superior to Anluohuaxian pills oninhibition of virus replication and improving the liver function.
2、HZKXF can elevate the levels of Th1and Th1/Th2, justify theimbalance of Th1/Th2,make the immune function develop towords Th1cell subsets dominant direction. Reduced glutathione tablets hasno effect on correcting the imbalance of Th1/Th2,
3、HZKXF can decrease the level of CD4~+CD25~+Foxp3~+Treg andTGF-β1, obstruct the bad circle of CD4~+CD25~+Foxp3~+Treg,TGF-β1andHSC, reverse the immune microenvironment in patients with liverfibrosis,promote the recovery of liver fibrosis.
4、HZKXF can elevate the levels of CD4~+, CD4~+/CD8+, decreasethe level of CD8+T lymphocyte.It can reduce the injury of livercells, promote the recovery of liver fibrosis in patients withchronic hepatitis B.At the same time,it can elevate the levels ofHBVcore18-27specific CTL and the function of sereting IFN-γ.Through correcting the imbalance of immune, HZKXF can controlthe HBV replication and improve the liver fibrosis causing by HBV.
观察海珠抗纤方治疗慢乙肝肝纤维化的疗效，检测该方治疗前后外周血T细胞亚群、CD4~+CD25~+Foxp3~+调节性T淋巴细胞、TGF-β1水平、HBV特异性细胞毒性T淋巴细胞的水平，探讨该方治疗慢性乙型肝炎肝纤维化的免疫机制，为中药从免疫的角度逆转肝纤维化提供理论基础，也为中医微观辩证提供依据。
方法：
1、将慢乙肝肝纤维化患者122例随机分为三组。治疗组62例，给予海珠抗纤方治疗，每日1剂，每天3次口服；对照1组60例，给予安络化纤丸治疗，6g/次，每日3次；对照2组给予还原型谷胱甘肽片治疗，每次0.4g口服，每天3次，三组疗程均为24周。观察各组治疗前后临床症状体征变化，检测各组治疗前后肝功能、血清HBVDNA水平、血清肝纤四项、肝脏Fibroscan弹性值的的变化。
2、将60例慢乙肝肝纤维化患者随机分为二组，治疗组30例，给予海珠抗纤方治疗，每日1剂，每天3次口服；对照组30例，给予还原型谷胱甘肽片治疗，每次0.4g口服，每天3次，两组疗程均为24周；选取健康体检成人30人为健康对照组。入选60例慢性乙型肝炎患者中有40例次行肝穿刺术，做肝组织病理学检查。检测各组治疗前后肝纤四项的水平；分别以细胞内表达IFN-γ作为Th1细胞亚群的标志，细胞内表达IL-4作为Th2细胞亚群的标志，通过流式细胞仪细胞内细胞因子染色法检测各组治疗前后Th1/Th2细胞亚群的变化，通过ELISA双抗夹心法检测各组治疗前后血清IFN-γ、IL-4水平变化；分析Th1/Th2细胞亚群与肝纤四项、肝组织病理纤维化分期之间的关系；观察海珠抗纤方对Th1/Th2细胞亚群的影响。
3、在第二部分研究的基础之上，通过流式细胞仪细胞内细胞因子染色法检测实验二中治疗组和对照组治疗前后血清CD4~+CD25~+Foxp3~+Treg细胞水平，通过ELISA双抗体夹心法检测治疗前后二组血清TGF-β1水平，进一步研究血清CD4~+CD25~+Foxp3~+Treg、TGF-β1水平与肝组织病理学及肝纤四项之间的关系，观察海珠抗纤方对慢乙肝肝纤维化CD4~+CD25~+Foxp3~+Treg、TGF-β1的影响。
4、应用HLA-A2作为过筛指标，经HLA抗体检测技术筛选HLA-A2（+）慢乙肝患者共62例。入选HLA-A2阳性慢乙肝患者随机分为二组。治疗组32例，给予海珠抗纤方治疗，每日1剂，每天3次口服；对照组30例，给予还原型谷胱甘肽片治疗，每次0.4g口服，每天3次，疗程均为24周。入选患者中有30例行肝穿术，做肝组织病理检查，检测治疗前后血清HBVDNA定量及肝纤四项水平；通过流式细胞仪免疫荧光法检测二组治疗前后CD4~+T淋巴细胞亚群、CD8+T淋巴细胞亚群、CD4~+/CD8+比值；通过流式细胞仪MHC-肽-五聚体法检测二组治疗前后HBV特异性细胞毒性T淋巴细胞水平及分泌IFN-γ功能的变化；分析T淋巴细胞亚群、HBV特异性细胞毒性T淋巴细胞与肝纤四项、肝组织纤维化分期之间的关系，观察海珠抗纤方对T淋巴细胞亚群和HBV特异性细胞毒性T淋巴细胞的影响。
结果：
1、海珠抗纤方治疗后慢乙肝肝纤维化患者临床症状及体征较治疗前明显好转(P＜0.05)，肝功能指标（ALT、AST、A/G、TBIL）较治疗前显著好转(P＜0.05)，其中治疗后ALT、AST改善上优于安络化纤丸组（对照1组），与西药护肝组（对照2组）比较无显著性差异(P＞0.05)；治疗后A/G比值改善上优于安络化纤丸组和西药护肝组（对照2组）(P＜0.05)。海珠抗纤方治疗后血清HBVDNA定量水平较治疗前显著下降(P＜0.05)；安络化纤丸组及西药护肝组治疗前后血清HBVDNA定量无明显改变(P＞0.05)。海珠抗纤方组及安络化纤丸组治疗后血清肝纤四项指标均较治疗前显著下降（P＜0.05），肝脏Fibroscan检测弹性值均较治疗前明显下降（P＜0.05），两组治疗后血清肝纤四项及Fibroscan弹性值比较无显著性差异(P＞0.05)；西药护肝组（对照2组）治疗前后肝纤四项及Fibroscan检测弹性值虽略有下降，但经统计学比较无显著性差异(P＞0.05)。
2、慢乙肝肝纤维化患者Th1、Th2细胞亚群水平及Th1/Th2比值均较健康对照组显著降低（P＜0.05），与健康对照组相比Th1下降更为明显，表现为Th2细胞亚群水平相对占优势。Th1细胞亚群水平与肝纤四项成负相关（r=－0.61，P＜0.05），并且随着肝脏纤维化分期的增加而下降；Th2细胞亚群水平与肝纤四项成正相关（r=0.43，P＜0.05），并且随着肝脏纤维化分期的增加而上升；Th1/Th2比值分别与肝纤四项、肝纤维化分期成负相关，相关系数分别为r=－0.52，P＜0.05，r=－0.68，P＜0.05。随着肝纤维化的进展，机体Th1细胞亚群水平逐渐降低，Th2细胞亚群水平逐渐升高，Th1/Th2比值逐渐降低，机体免疫应答出现由Th1向Th2漂移的现象。海珠抗纤方治疗后Th1细胞亚群水平及Th1/Th2比值均明显升高（P＜0.05），而对照组治疗前后Th1、Th2细胞亚群水平及Th1/Th2比值均无显著性改变（P＞0.05）。
3、慢乙肝肝纤维化患者治疗前血清肝纤四项、TGF-β1及CD4~+CD25~+Foxp3~+Treg细胞亚群水平较健康对照组明显升高（P＜0.05），治疗组治疗后血清肝纤四项、TGF-β1及CD4~+CD25~+Foxp3~+Treg细胞亚群水平较治疗前显著降低（P＜0.05）；对照组治疗前后血清肝纤四项、TGF-β1及CD4~+CD25~+Foxp3~+Treg细胞亚群水平无明显改变（P＞0.05）。血清TGF-β1水平与肝纤四项成正相关，（相关系数分别为:rHA=0.82, P＜0.05；rLN=0.69, P＜0.05；rPCⅢ=0.72,P＜0.05；rC-Ⅳ=0.65, P＜0.05）；血清TGF-β1水平与肝脏病理分期成正相关（r=0.84，P＜0.05），血清CD4~+CD25~+Foxp3~+Treg细胞亚群水平与TGF-β1水平及肝脏病理分期成显著正相关（相关系数为r=0.58，P＜0.05；r=0.55，P＜0.05）。
4、慢乙肝肝纤维化患者治疗前CD4~+T比例及CD4~+/CD8+比值较健康对照组显著下降(P＜0.05)，CD8+T比例明显升高（P＜0.05)，血清CD4~+T淋巴细胞比例及CD4~+/CD8+比值与肝脏纤维化病理分期成负相关（相关系数分别为rCD4~+=－0.53，P＜0.05；rCD4~+/CD8+=－0.59，P＜0.05）；血清CD8+T淋巴细胞比例与肝脏纤维化病理分期成正相关（rCD8+=0.42，P＜0.05）。随着肝纤维化程度的加重，CD4~+T淋巴细胞比例及CD4~+/CD8+比值逐渐下降，CD8+T淋巴细胞比例逐渐上升，T细胞亚群的紊乱逐渐加重。海珠抗纤方治疗后血清CD4~+T淋巴细胞比例、CD4~+/CD8+比值、HBVcore18-27特异性细胞毒性T淋巴细胞频数及产生IFN-γ的频数较治疗前显著提高（P＜0.05）；CD8+T淋巴细胞比例、血清HBVDNA水平较治疗前显著下降（P＜0.05）；对照组治疗治疗前后T淋巴细胞亚群、HBVDNA水平、HBVcore18-27特异性细胞毒性T淋巴细胞频数及产生IFN-γ的频数均无显著性差异（P＞0.05）。
结论：
1、海珠抗纤方能够改善慢性乙型肝炎肝纤维化患者的临床症状和体征，改善肝功能，升高A/G比值，降低血清HBVDNA含量，降低血清肝纤四项和改善肝脏硬度，其抗纤维化的作用与安络化纤丸相比无显著性差异，但在抑制病毒复制和改善肝功能指标上优于安络化纤丸。
2、海珠抗纤方可以升高慢乙肝肝纤维化患者血清Th1细胞亚群水平及Th1/Th2比值，从而纠正Th1/Th2失衡，使机体免疫功能向Th1细胞亚群占优势的方向逆转。还原性谷胱甘肽片对纠正Th1/Th2失衡无明显作用。
3、海珠抗纤方能显著降低慢乙肝肝纤维化患者血清CD4~+CD25~+Foxp3~+Treg及TGF-β1水平，对血清CD4~+CD25~+Foxp3~+Treg及TGF-β1水平有一定调节作用，可以阻断CD4~+CD25~+Foxp3~+Treg-TGF-β1-HSC三者之间的循环反应，逆转肝纤维化患者HSC的免疫微环境，促进肝纤维化的恢复。
4、海珠抗纤方通过提高CD4~+T淋巴细胞比例、CD4~+/CD8+比值，降低CD8+T淋巴细胞比例，从而减轻肝细胞损伤，促进慢性乙型肝炎肝纤维化的恢复。同时可以提高慢乙肝肝纤维化患者HBV抗原特异性CTL的数量及分泌IFN-γ的功能。海珠抗纤方通过纠正慢乙肝肝纤维化患者的免疫失衡既有利于控制HBV病毒复制，也有利于逆转乙型肝炎肝纤维化。
Objective:Observe the clinical effect of HZKXF on liver fibrosisin patients of chronic hepatitis B.Detect the serum level of Th1/Th2,CD4~+CD25~+Foxp3~+Treg, TGF-β1,T lymphocyte subsets and HBV specificCTL.Probe into the immunologic mechanism of HZKXF on fibrosis.
Methods:
1、122patients of chronic hepatitis B were divided into3groupsrandomly.The treatment group was treated by HZKXF(everyday1agent,3times a day).The control group1was treated by Anluohuaxianpills (6grams evevy time,3times a day). Control group2wastreated by reduced glutathione tablets(0.4grams evevy time,3times a day).The courses of treatment were all24weeks in everygroup.Detect the level of liver function,HBVDNA quantity andHA,LN,PCⅢ,C-Ⅳ,liver elasticity number before and after thetreatment.Observe the clinical effect of HZKXF on liver fibrosis.
2、60patients of chronic hepatitis B were divided into2groupswith random.The treatment group was treated by HZKXF(everyday1agent,3times a day).The control group was treated by reducedglutathione tablets(0.4grams evevy time,3times a day). Thecourses of treatment were both24weeks.30healthy people were thehealthy control group.There are40times liver puncture among60patients. Detect the level of HA,LN,PCⅢ,C-Ⅳ, the serum level ofIFN-,IL-4,Th1/Th2before and after the treatment.Analyse the correlativity relations between Th1/Th2, serum hepatic fibrosisindexes and the stage of liver fibrosis. Observe the effect of HZKXFon Th1/Th2.
3、Based on the second experiment,using the flow cytometryintracellular cytokine staining method to dectect the level ofCD4~+CD25~+Foxp3~+Treg in serum before and after the treatment.Usingthe ELISA double antibody sandwich method to dectect the level ofTGF-β1in serum before and after the treatment. Analyse the corr-elativity relations between CD4~+CD25~+Foxp3~+Treg,TGF-β1, serumhepatic fibrosis indexes and the stage of liver fibrosis. Observethe effect of HZKXF on CD4~+CD25~+Foxp3~+Treg and TGF-β1.
4、Application of HLA-A2as a screening index，62patients ofHLA-A2antibody(+) were divided into2groups randomly in patientswith chronic hepatitis B. There were32patients in the treatmentgroup.The treatment group was treated by HZKXF (everyday1agent,3times a day). There were30patients in the control group.Thecontrol group was treated by reduced glutathione tablets(0.4gramsevevy time,3times a day).The courses of treatment were all24weeksin every group.Using flow cytometry to dectect the level of CD4~+,CD8+and CD4~+/CD8+in serum before and after treatment.Using MHC-HBVcore18-27-pentmer to dectect the level of HBV specific CTL and the itsfunction of secreting IFN-γin serum before and after the treatment.Analyse the correlativity relations between T lymphocyte subsets,HBV specific CTL,HBVDNA quantity serum hepatic fibrosis indexes andthe stage of liver fibrosis.
Results:
1、The level of liver function was improved obviously after HXKXF treatment(P＜0.05). The level of ALT and AST were improvedmore obviously in the treatment group than in the control group1.There were no significant differences on the level of ALT and ASTbetween the treatment group and the control group2after thetreatment(P＞0.05).The level of A/G was improved more obviously inthe treatment group than in the control group1and control group.
2、The level of HBVDNA quantity was decreased more obviouslyin the treatment group than in the countrol group1and countrolgroup2. There were no significant differences on the level ofHBVDNA quantity both in the control group1and control group2afterthe treatment(P＞0.05). The level of liver fibrosis indexes inserum and liver elasticity numbers detecting with Fibroscan weredecreased obviously after HXKXF and Anluohuaxian pills treatment(P＜0.05).There were no significant differences between the2groupson the level of liver fibrosis indexes in serum and liver elasticitynumbers(P＞0.05).There were no significant differences on thelevel of liver fibrosis indexes in serum and liver elasticitynumbers after the treatment in the control group2(P＞0.05).
3、The level of Th1、Th2and Th1/Th2decreased obviously comparedwith the healthy control group(P＜0.05).The level of Th1decreasedmore obviously than the level of Th2. The level of Th1negativelycorrelated with the liver fibrosis indexes in serum（r=－0.71，P＜0.05）,it declined with the stage of liver fibrosis rised.Th2positively correlated with the liver fibrosis indexes in serum（r=0.58，P＜0.05）,it elevated the stage of liver fibrosis rised.Th1/Th2negatively correlated with the liver fibrosis indexes inserum（r=－0.82，P＜0.05） and the stage of liver fibrosis(r=－0.84，P＜0.05).The level of Th1and Th1/Th2elevated after HZKXF treatment（P＜0.05）.The level of Th2decreased after HZKXFtreatment（P＜0.05）. There were no significant differences on thelevel of Th1, Th2and Th1/Th2after the treatment in the controlgroup (P＞0.05).
4、 The level of the liver fibrosis indexes,TGF-β1andCD4~+CD25~+Foxp3~+Treg in serum rised obviously compared with thehealthy control group(P＜0.05). The level of the liver fibrosisindexes,TGF-β1and CD4~+CD25~+Foxp3~+Treg in serum decreased afterHZKXF treatment(P＜0.05). There were no significant differences onthe level of the liver fibrosis indexes,TGF-β1andCD4~+CD25~+Foxp3~+Treg in serum after the treatment in the control group(P＞0.05).The level of TGF-β1positively correlated with the liverfibrosis indexes in serum（rHA=0.56, P＜0.05；rLN=0.58, P＜0.05；rPC Ⅲ=0.56,P＜0.05；rC-Ⅳ=0.48, P＜0.05）. The level of TGF-β1positively correlated with the stage of liver fibrosis（r=0.51，P＜0.05）.The level of CD4~+CD25~+Foxp3~+Treg positively correlatedwith the level of TGF-β1in serum.
5、The level of CD4~+T and CD4~+/CD8+in serum decreased,the levelof CD8+rised obviously compared with the healthy control groupbefore the treatment(P＜0.05).The level of CD4~+T and CD4~+/CD8+negatively correlated with the stage of liver fibrosis(rCD4~+=－0.53，P＜0.05；rCD4~+/CD8+=－0.59，P＜0.05）. The level of CD8+positivelycorrelated with the stage of liver fibrosis（rCD8+=0.55，P＜0.05）.Thelevel of CD4~+, CD4~+/CD8+, HBVcore18-27specific CTL and seretingIFN-γ improved after HXKXF treatment（P＜0.05）.The level of CD8+and HBVDNA quantity decreased after HXKXF treatment（P＜0.05）.There were no significant differences on the level of T lymphocyte subsets, HBVcore18-27specific CTL and sereting IFN-γin serumafter the treatment in the control group (P＞0.05).
Conclusion:
1、HZKXF can improved the liver function,decrease the levelof HBVDNA quantity,liver fibrosis indexes, liver elasticitynumbers. There were no significant differences on anti-hepaticfibrosis effects(P＞0.05).Itis superior to Anluohuaxian pills oninhibition of virus replication and improving the liver function.
2、HZKXF can elevate the levels of Th1and Th1/Th2, justify theimbalance of Th1/Th2,make the immune function develop towords Th1cell subsets dominant direction. Reduced glutathione tablets hasno effect on correcting the imbalance of Th1/Th2,
3、HZKXF can decrease the level of CD4~+CD25~+Foxp3~+Treg andTGF-β1, obstruct the bad circle of CD4~+CD25~+Foxp3~+Treg,TGF-β1andHSC, reverse the immune microenvironment in patients with liverfibrosis,promote the recovery of liver fibrosis.
4、HZKXF can elevate the levels of CD4~+, CD4~+/CD8+, decreasethe level of CD8+T lymphocyte.It can reduce the injury of livercells, promote the recovery of liver fibrosis in patients withchronic hepatitis B.At the same time,it can elevate the levels ofHBVcore18-27specific CTL and the function of sereting IFN-γ.Through correcting the imbalance of immune, HZKXF can controlthe HBV replication and improve the liver fibrosis causing by HBV.
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