益气解毒法对大鼠肝干细胞增殖及肝脏再生影响的机制研究
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摘要
1.目的
西洋参、牛黄具有益气养阴、清热解毒之功效,符合“益气解毒法”的基本原则。将两药有效成分配伍组合,观察“益气解毒法”对大鼠肝干细胞系诱导分化,增殖的影响和对大鼠肝再生的效果,探索益气解毒法治疗慢性肝病的作用原理,为进一步研究治则治法提供新思路,也为临床推广应用提供理论与实验依据。
2.方法
2.1分组
正常组,手术组(不灌服2-AAF),模型组,中药水煎组5%组、10%组、20%组,中药成分组5%组、10%组、20%组。
2.2造模
选常用的solt-Farbar造模方法,稍作改进。随机分组分笼饲养1周后,胃管灌服2-AAF(按10mg/kg鼠重),每日1次,连续4d,第5日戊巴比妥钠(50mg/kg)腹腔注射麻醉下行肝三分之二(左叶和中叶)切除(当日停灌2-AAF),肝叶切除方法,仰卧固定,常规备皮消毒,取上腹正中切口入腹,游离左叶和中叶动脉、结扎,待左、中叶肝颜色变黄,切除左叶和中叶,结扎动脉、静脉和胆管,无出血、胆瘘关腹。手术当天不给药。第6日连续灌服2-AAF1周,再分别中药灌服(剂量为整体模型动物的有效剂量),并设正常对照组,对照组给予生理盐水灌喂。于手术后第13、16、20、25天分别取3只大鼠,戊巴比妥钠(50mg/kg)腹腔注射麻醉后,打开腹腔,暴露肝脏,无菌腹主动脉采血,分离血清置-30℃保存待检。迅速剪取肝脏10-20g快速液氮冷冻过夜后置于-80℃冷存备检,并取再生肝组织和右叶肝,10%甲醛浓度固定,常规石蜡包埋,5umm连续切片备用。
2.3观察指标的检测
肝干细胞(卵圆细胞)增殖情况：肝卵圆细胞核运用计算机图像分析系统进行卵圆细胞、肝细胞标志物计数定量分析比较。
原位杂交法检测肝组织TGF-α、TGF-β1mRNA表达：制备TGF-α RNA和TGF-α RNA探针,TGF-α采用DNA-RNA原位杂交法,TGF-β1采用RNA-RNA杂交法,按试剂盒说明操作。
肝组织TGF-β1定量检测：取0.1g液氮冻存肝组织匀浆,提取总RNA,运用RT-PCR联合DotBlot法定量检测TGF-β1。
再生肝细胞分裂指数(MI)：取再生肝组织10%甲醛固定,常规石蜡包埋,苏木素-伊红染色,油镜下观察计数肝细胞核总数和肝细胞核分裂数,以下公式计算：MI%=肝细胞核有丝分裂象(个)/肝细胞核总数(个)x100%。
肝组织EGFR、TGF-βR2、HGFR受体蛋白的表达：取肝组织5um连续切片,采用SABC免疫组织化学法检测上述受体,用已知的EGFR、TGF-β R2、HGFR阳性大鼠肝组织作阳性对照,用PBS代替一抗作空白阴性对照。
一般指标的观察：如大鼠体重、毛发、粪便、分泌物、活动状况等,血肝功能生化检测,肝组织切片组织学观察。
3.结果
3.1成分组与水煎组PCNA、波形蛋白、肝细胞CK8、CK18表达均大于手术组与正常组(P<0.05),表明成功建立了肝卵圆细胞模型。
3.2成分组与水煎组TGF-β1表达均大于模型组与手术组(P<0.05),表明益气解毒法能通过TGF-β1干预了肝干细胞诱导分化。
3.3成分组与水煎组TGF-α、TGF-β1mRNA表达均优于模型组与手术组(P<0.05),表明益气解毒法能通过TGF-α、TGF-β1mRNA干预了肝干细胞诱导分化。
3.4成分组与水煎组EGFR、TGF-βR2、HGFR受体蛋白表达均优于模型组与手术组(P<0.05),表明益气解毒法能通过EGFR、TGF-βR2、HGFR受体蛋白干预了肝干细胞诱导分化。
3.5成分组与水煎组在改善大鼠一般情况方面均优于模型组与手术组(P<0.05),表明益气解毒法能调节肝干细胞诱导分化。
4、结论
4.1.2-AAF联合肝损伤较单纯肝损伤造模方法好,大鼠死亡率低。
4.2.益气解毒法能通过调节TGF-β1干预肝干细胞诱导、增值和分化。
4.3.益气解毒法能通过调节TGF-a、TGF-β1mRNA干预肝干细胞诱导、增值和分化。
4.4.益气解毒法能通过调节EGFR、TGF-βR2、HGFR干预肝干细胞诱导、增值和分化。
4.5.益气解毒法能改善大鼠一般情况,提高其生存质量,改善其肝功能。
1. Objective
American ginseng, the Bezoar have the Yiqiyangyin Qingrejiedu function, to conform Yiqijiedu "basic principles. Compatibility combination of the active ingredients of the two drugs to observe the impact and effect of liver regeneration "Yiqijiedu Law on rat liver stem cell lines induced differentiation, proliferation, to explore the role of principle Yiqijiedu treatment of chronic liver disease, in order to further study therapeutic principle to provide new ideas, and also provide a theoretical and experimental basis for clinical application.
2Methods
2.1Grouping
Normal group, operation group (orally2-AAF), model group, Chinese medicine decoction group5%group,10%of the group,20%of the group, Chinese medicines group group5%,10%group,20%of the group.
2.2modeling
Selected the frequently used the solt-Farbar modeling method to make some improvements. Randomized housed separately one week after the gastric tube fed AAF (10mg/kg mouse weight), once a day, four consecutive days, on the5th of sodium pentobarbital (50mg/kg) by intraperitoneal injection anesthesia for liver three two-thirds (left lobe and middle) resection (the day stopping irrigation AAF) hepatectomy method, supine fixed conventional skin preparation disinfection, to take abdominal incision Rufu, free left and middle lobes artery ligation, to be left mid-liver-yellowing, resection of the left and middle lobes, ligation of the artery, vein and bile duct, no bleeding, biliary fistula in the abdomen was closed. The day of surgery is not administered. The6th consecutive irrigation serving AAF1weeks, respectively Chinese medicine orally (dose, the effective dose of the overall model animal), and the normal control group, the control group received saline intragastrically.13,16,20,25days after surgery were taken three rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50mg/kg), the abdominal cavity was opened, exposing the liver, sterile abdominal aortic blood, separation of the serum set stored at-30℃for inspection. Quickly clipping liver10-20g quick frozen in liquid nitrogen overnight at-80℃cold kept examination and take the regeneration of liver tissue and the right lobe of the liver, a fixed concentration of10%formaldehyde, embedded in paraffin,5um serial sections alternate.
2.3OUTCOME MEASURES detection
Liver stem cells (oval cell) proliferation:hepatic oval nucleus use computer image analysis system of oval cells, liver cells count quantitative analysis and comparison. In situ hybridization detection of liver tissue TGF-a, TGF-β1mRNA expression:Preparation of TGF-aCDNA and the TGF-β1RNA probe, TGF-a RNA-RNA hybridization using DNA-RNA in situ hybridization, TGF-β1, press The kit instructions.
Quantitative detection of TGF-β1in liver tissue:Take0.1g liquid nitrogen frozen liver tissue homogenates, extraction of total RNA, detected using RT-PCR the United DotBlot statutory amount of TGF-β1.
Regenerating liver cell division index (MI):take regeneration in liver tissue fixed in10%formalin, embedded in paraffin, hematoxylin-eosin staining, oil microscope to count the total number of liver cell nuclei and hepatocyte nuclear division of the following formula:MI%=hepatocyte nuclear mitotic (a)/(a) x100%the total number of hepatocyte nuclear.
Liver tissue EGFR, of TGF-βR2, and HGFR receptor protein:liver tissue5um consecutive slices by SABC immunohistochemical method to detect the receptor, known EGFR of TGF-βR1, R2, and HGFR positive rat liver tissue as a positive control for blank negative control antibody was replaced with PBS.
General observation of indicators:body weight of rats, hair, feces, secretions, activity status, blood liver function biochemical detection, liver tissue biopsy tissue observations.
3Results
3.1component group decoction group, PCNA, vimentin, liver cells CK8, CK18expression is greater than the surgical group and the normal group (P<0.05), indicating a successful hepatic oval cell model.
3.2component group decoction group, the expression of TGF-p is greater than the model group and the surgery group (P<0.05), and through TGF-β intervention liver stem cells induced to differentiate that Yiqijiedu law.
3.3component group decoction group the TGF-a, TGF-β1mRNA expression are better than the model group and the surgery group (P<0.05), through the TGF-a that Yiqijiedu law, TGF-β1mRNA intervention liver stem cells induced to differentiate.
3.4component group with the of water decoction group of EGFR of TGF-βR1, R2, HGFR receptor protein are better than the model group and the surgery group (P<0.05), indicating the Yiqijiedu law through EGFR the TGF-βR2, HGFR, The receptor proteins intervention liver stem cells induced to differentiate.
3.5component group decoction group are better than in the improvement of the general situation of the rat model with operation group (P<0.05), that Yiqijiedu law can regulate liver stem cells induced to differentiate.
4Conclusion
4.1.2-AAF combined liver injury than a simple modeling of liver injury, the the rat mortality rate is low.
4.2Yiqijiedu by regulating TGF-β intervention induced liver stem cells, proliferation and differentiation.
4.3Yiqijiedu through regulation of TGF-a, TGF-β1mRNA intervention liver stem cells induced proliferation and differentiation.
4.4Yiqijiedu through regulation of EGFR, the TGF-βR2, HGFR the intervention liver stem cells induced proliferation and differentiation.
4.5Yiqijiedu to improve the general situation of the rat, to improve their quality of life, improve liver function.
西洋参、牛黄具有益气养阴、清热解毒之功效,符合“益气解毒法”的基本原则。将两药有效成分配伍组合,观察“益气解毒法”对大鼠肝干细胞系诱导分化,增殖的影响和对大鼠肝再生的效果,探索益气解毒法治疗慢性肝病的作用原理,为进一步研究治则治法提供新思路,也为临床推广应用提供理论与实验依据。
2.方法
2.1分组
正常组,手术组(不灌服2-AAF),模型组,中药水煎组5%组、10%组、20%组,中药成分组5%组、10%组、20%组。
2.2造模
选常用的solt-Farbar造模方法,稍作改进。随机分组分笼饲养1周后,胃管灌服2-AAF(按10mg/kg鼠重),每日1次,连续4d,第5日戊巴比妥钠(50mg/kg)腹腔注射麻醉下行肝三分之二(左叶和中叶)切除(当日停灌2-AAF),肝叶切除方法,仰卧固定,常规备皮消毒,取上腹正中切口入腹,游离左叶和中叶动脉、结扎,待左、中叶肝颜色变黄,切除左叶和中叶,结扎动脉、静脉和胆管,无出血、胆瘘关腹。手术当天不给药。第6日连续灌服2-AAF1周,再分别中药灌服(剂量为整体模型动物的有效剂量),并设正常对照组,对照组给予生理盐水灌喂。于手术后第13、16、20、25天分别取3只大鼠,戊巴比妥钠(50mg/kg)腹腔注射麻醉后,打开腹腔,暴露肝脏,无菌腹主动脉采血,分离血清置-30℃保存待检。迅速剪取肝脏10-20g快速液氮冷冻过夜后置于-80℃冷存备检,并取再生肝组织和右叶肝,10%甲醛浓度固定,常规石蜡包埋,5umm连续切片备用。
2.3观察指标的检测
肝干细胞(卵圆细胞)增殖情况：肝卵圆细胞核运用计算机图像分析系统进行卵圆细胞、肝细胞标志物计数定量分析比较。
原位杂交法检测肝组织TGF-α、TGF-β1mRNA表达：制备TGF-α RNA和TGF-α RNA探针,TGF-α采用DNA-RNA原位杂交法,TGF-β1采用RNA-RNA杂交法,按试剂盒说明操作。
肝组织TGF-β1定量检测：取0.1g液氮冻存肝组织匀浆,提取总RNA,运用RT-PCR联合DotBlot法定量检测TGF-β1。
再生肝细胞分裂指数(MI)：取再生肝组织10%甲醛固定,常规石蜡包埋,苏木素-伊红染色,油镜下观察计数肝细胞核总数和肝细胞核分裂数,以下公式计算：MI%=肝细胞核有丝分裂象(个)/肝细胞核总数(个)x100%。
肝组织EGFR、TGF-βR2、HGFR受体蛋白的表达：取肝组织5um连续切片,采用SABC免疫组织化学法检测上述受体,用已知的EGFR、TGF-β R2、HGFR阳性大鼠肝组织作阳性对照,用PBS代替一抗作空白阴性对照。
一般指标的观察：如大鼠体重、毛发、粪便、分泌物、活动状况等,血肝功能生化检测,肝组织切片组织学观察。
3.结果
3.1成分组与水煎组PCNA、波形蛋白、肝细胞CK8、CK18表达均大于手术组与正常组(P<0.05),表明成功建立了肝卵圆细胞模型。
3.2成分组与水煎组TGF-β1表达均大于模型组与手术组(P<0.05),表明益气解毒法能通过TGF-β1干预了肝干细胞诱导分化。
3.3成分组与水煎组TGF-α、TGF-β1mRNA表达均优于模型组与手术组(P<0.05),表明益气解毒法能通过TGF-α、TGF-β1mRNA干预了肝干细胞诱导分化。
3.4成分组与水煎组EGFR、TGF-βR2、HGFR受体蛋白表达均优于模型组与手术组(P<0.05),表明益气解毒法能通过EGFR、TGF-βR2、HGFR受体蛋白干预了肝干细胞诱导分化。
3.5成分组与水煎组在改善大鼠一般情况方面均优于模型组与手术组(P<0.05),表明益气解毒法能调节肝干细胞诱导分化。
4、结论
4.1.2-AAF联合肝损伤较单纯肝损伤造模方法好,大鼠死亡率低。
4.2.益气解毒法能通过调节TGF-β1干预肝干细胞诱导、增值和分化。
4.3.益气解毒法能通过调节TGF-a、TGF-β1mRNA干预肝干细胞诱导、增值和分化。
4.4.益气解毒法能通过调节EGFR、TGF-βR2、HGFR干预肝干细胞诱导、增值和分化。
4.5.益气解毒法能改善大鼠一般情况,提高其生存质量,改善其肝功能。
1. Objective
American ginseng, the Bezoar have the Yiqiyangyin Qingrejiedu function, to conform Yiqijiedu "basic principles. Compatibility combination of the active ingredients of the two drugs to observe the impact and effect of liver regeneration "Yiqijiedu Law on rat liver stem cell lines induced differentiation, proliferation, to explore the role of principle Yiqijiedu treatment of chronic liver disease, in order to further study therapeutic principle to provide new ideas, and also provide a theoretical and experimental basis for clinical application.
2Methods
2.1Grouping
Normal group, operation group (orally2-AAF), model group, Chinese medicine decoction group5%group,10%of the group,20%of the group, Chinese medicines group group5%,10%group,20%of the group.
2.2modeling
Selected the frequently used the solt-Farbar modeling method to make some improvements. Randomized housed separately one week after the gastric tube fed AAF (10mg/kg mouse weight), once a day, four consecutive days, on the5th of sodium pentobarbital (50mg/kg) by intraperitoneal injection anesthesia for liver three two-thirds (left lobe and middle) resection (the day stopping irrigation AAF) hepatectomy method, supine fixed conventional skin preparation disinfection, to take abdominal incision Rufu, free left and middle lobes artery ligation, to be left mid-liver-yellowing, resection of the left and middle lobes, ligation of the artery, vein and bile duct, no bleeding, biliary fistula in the abdomen was closed. The day of surgery is not administered. The6th consecutive irrigation serving AAF1weeks, respectively Chinese medicine orally (dose, the effective dose of the overall model animal), and the normal control group, the control group received saline intragastrically.13,16,20,25days after surgery were taken three rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50mg/kg), the abdominal cavity was opened, exposing the liver, sterile abdominal aortic blood, separation of the serum set stored at-30℃for inspection. Quickly clipping liver10-20g quick frozen in liquid nitrogen overnight at-80℃cold kept examination and take the regeneration of liver tissue and the right lobe of the liver, a fixed concentration of10%formaldehyde, embedded in paraffin,5um serial sections alternate.
2.3OUTCOME MEASURES detection
Liver stem cells (oval cell) proliferation:hepatic oval nucleus use computer image analysis system of oval cells, liver cells count quantitative analysis and comparison. In situ hybridization detection of liver tissue TGF-a, TGF-β1mRNA expression:Preparation of TGF-aCDNA and the TGF-β1RNA probe, TGF-a RNA-RNA hybridization using DNA-RNA in situ hybridization, TGF-β1, press The kit instructions.
Quantitative detection of TGF-β1in liver tissue:Take0.1g liquid nitrogen frozen liver tissue homogenates, extraction of total RNA, detected using RT-PCR the United DotBlot statutory amount of TGF-β1.
Regenerating liver cell division index (MI):take regeneration in liver tissue fixed in10%formalin, embedded in paraffin, hematoxylin-eosin staining, oil microscope to count the total number of liver cell nuclei and hepatocyte nuclear division of the following formula:MI%=hepatocyte nuclear mitotic (a)/(a) x100%the total number of hepatocyte nuclear.
Liver tissue EGFR, of TGF-βR2, and HGFR receptor protein:liver tissue5um consecutive slices by SABC immunohistochemical method to detect the receptor, known EGFR of TGF-βR1, R2, and HGFR positive rat liver tissue as a positive control for blank negative control antibody was replaced with PBS.
General observation of indicators:body weight of rats, hair, feces, secretions, activity status, blood liver function biochemical detection, liver tissue biopsy tissue observations.
3Results
3.1component group decoction group, PCNA, vimentin, liver cells CK8, CK18expression is greater than the surgical group and the normal group (P<0.05), indicating a successful hepatic oval cell model.
3.2component group decoction group, the expression of TGF-p is greater than the model group and the surgery group (P<0.05), and through TGF-β intervention liver stem cells induced to differentiate that Yiqijiedu law.
3.3component group decoction group the TGF-a, TGF-β1mRNA expression are better than the model group and the surgery group (P<0.05), through the TGF-a that Yiqijiedu law, TGF-β1mRNA intervention liver stem cells induced to differentiate.
3.4component group with the of water decoction group of EGFR of TGF-βR1, R2, HGFR receptor protein are better than the model group and the surgery group (P<0.05), indicating the Yiqijiedu law through EGFR the TGF-βR2, HGFR, The receptor proteins intervention liver stem cells induced to differentiate.
3.5component group decoction group are better than in the improvement of the general situation of the rat model with operation group (P<0.05), that Yiqijiedu law can regulate liver stem cells induced to differentiate.
4Conclusion
4.1.2-AAF combined liver injury than a simple modeling of liver injury, the the rat mortality rate is low.
4.2Yiqijiedu by regulating TGF-β intervention induced liver stem cells, proliferation and differentiation.
4.3Yiqijiedu through regulation of TGF-a, TGF-β1mRNA intervention liver stem cells induced proliferation and differentiation.
4.4Yiqijiedu through regulation of EGFR, the TGF-βR2, HGFR the intervention liver stem cells induced proliferation and differentiation.
4.5Yiqijiedu to improve the general situation of the rat, to improve their quality of life, improve liver function.
引文
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