龟板有效成分对胎鼠表皮干细胞凋亡的影响
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摘要
一、研究目的
干细胞是目前生命科学研究的重点和热点,其中表皮干细胞是解决各种皮肤问题的重要途径。表皮干细胞(epidermal stem cells, ESCs)具有易分离、易扩增及体外操作简便等特点,所以在临床上有较广泛的应用前景。紫外辐射和失营养是造成皮肤损伤的重要因素,因此,寻找安全有效的抗表皮干细胞紫外辐射凋亡、失营养凋亡的方法是当前研究的重要方向。
龟板具有滋阴潜阳,补肾壮骨,养血补心等功效。本研究课题组早前实验发现,龟板有效成分PTE(龟板乙酸乙酯部位提取物,简称2B)在早期可以促进骨髓间充质干细胞(mesenchvmal stem cells, MSCs)的增殖,继续培养,则促进了MSCs向成骨细胞的分化。另外还发现龟板对帕金森病大鼠多巴胺能神经元具有抗凋亡的作用。龟板作为一种补肾中药是否也可以促进ESCs增殖,对抗损伤的ESCs凋亡,及其相关作用机制是什么,目前还不清楚,因此本实验希望通过龟板有效成分对ESCs影响的研究,从而为临床医学皮肤损伤修复及护肤美容产品开发提供一种可能的新的途径。
二、研究方法
(1)表皮干细胞的培养与纯化
本实验取用孕两周Sprague-Dawley (SD)大鼠腹中胎鼠的背部皮肤,剪碎后加入胰酶消化,并收集细胞进行培养。收集第Ⅰ至Ⅳ代细胞,采用ESCs的标记物角蛋白19(keratin19, K19)和β1整合素(β1-integrin)进行双标鉴定,检测所培养细胞的性质和纯度,选取纯度最高的第Ⅲ代表皮干细胞进行以下实验。(2)龟板有效成分抗表皮干细胞紫外线损伤凋亡实验
确定紫外损伤模型：将细胞种入6孔板,培养24h后,吸出孔内的培养基,并用D-Hank's液清洗,按一定损伤量(100mJ/cm2)、不同损伤时间分组,分为三组,分别紫外照射5min、15min、30min, D-Hank's液清洗,加入培养基继续培养10h、20h、36h,之后进行流式细胞术AnnexinⅤ/PI双标检测各组凋亡率,依据凋亡率结果确定：紫外损伤15min,继续培养20h可获得稳定的凋亡模型。
药物干预：将细胞分为正常组、模型组、药物组。药物组又分为2B组、S6组(十八酸乙酯)、S8组(十四酸甾醇酯)、S9组(4-甾酮)。正常组无特殊处理,模型组方法同前,药物组与模型组不同的是,损伤后所加培养基中添加2B、S6、S8或S9,通过流式细胞术测定凋亡率来比较龟板有效成分不同部位抗凋亡的效果；免疫组化法观察各组中p53、caspase3和bcl-2的阳性表达情况；蛋白免疫印迹法检测socs7、nck、p53、caspase3等蛋白的表达水平。
(3)龟板有效成分抗表皮干细胞无血清损伤凋亡实验
将细胞种入96孔板,培养24h后,正常组不做特殊处理,吸出模型组、药物组孔内的培养基,并用D-Hank's液清洗,模型组加入无血清培养基,药物组加入含龟板有效成分的无血清培养基,分别培养24h、48h、72h,MTT法检测各组光密度值(OD值),确立最佳药效时段。通过流式细胞术检测最佳时段中各种药物组细胞凋亡率,比较各药物组抗凋亡的效果。免疫印迹检测caspase3和bcl-2的表达情况。
三、实验结果
(1)表皮干细胞的培养与纯化
流式细胞术检测第Ⅰ至Ⅳ代细胞K19和β1整合素双标阳性百分率平均值分别是89.75%、94.55%、96.57%、78.75%。结果提示：胎鼠背部皮肤中ESCs含量丰富,第Ⅲ代表皮干细胞K19和β1整合素的双标阳性率明显高于第Ⅰ、Ⅱ、Ⅳ代细胞,说明第Ⅲ代表皮干细胞纯度最高。
(2)龟板有效成分抗表皮干细胞紫外线损伤凋亡实验
表皮干细胞在100mJ/cm2紫外损伤量下照射15min,再培养20h,与其他损伤时间组(5min组、30min组)、其他损伤后培养时间组(12h组、36h组)相比,细胞正常率较高,凋亡率较高,死亡率较低,可作为紫外辐射凋亡模型。免疫组化结果显示：bcl-2在细胞浆和细胞核均有表达,caspase3表达于细胞浆中,P53主要表达于细胞核内。龟板有效成分各组中bcl-2阳性细胞数多于模型组,而caspase3、p53阳性细胞数少于模型组。免疫印迹结果显示PTE中S8、S9组明显降低socs7、nck、p53、caspase3等蛋白的表达水平。
(3)龟板有效成分抗表皮干细胞无血清损伤凋亡实验
MTT结果表明,PTE作用于无血清培养的表皮干细胞,48h时0D值高于24h组、72h组；流式检测结果表明48h时各药物组与模型组相比,有较好的抗凋亡作用；免疫印迹显示各药物组均上调bcl-2,下调caspase3,其中以2B效果最明显。
四、结论
(1)胎鼠背部皮肤ESCs含量丰富；细胞培养至第Ⅲ代, ESCs纯度最高。
(2)龟板有效成分抗表皮干细胞紫外线损伤凋亡,其中S8、S9抗凋亡效果最明显。
(3)龟板有效成分抗表皮干细胞无血清损伤凋亡,以2B效果最明显。
Objective
Stem cells is one of the most attracting fields in the life sciences in the recent decades and Epidermal Stem Cells (ESCs) is the significant approach to deal with various kinds of skin problems. ESCs is one of stem cells, which has the characteristic that it is easy to separate, proliferate and culture in vitro, has widespreadly applying prospect in clinic. Ultra-Violet (UV) radiation and nutriment deficiency are the significant causes to the skin damage, so it is the most important problem that how to find an safe and effective method to inhibit ESCs apoptosis induced by Ultra-Violet radiation and nutriment deficiency. Based on Traditional Chinese Medicine theory that "the kidney being the origin of congenital constitution, controlling growth and reproduction", we hope to promote ESCs proliferation. and resist apoptosis with the Chinese Medicine PlastrumTestudinis (PT) which can invigorate the kidney.
Traditional Chinese Medicine holds that PT has the efficacy of nourishing liver and kidney, so it is often used as an important Traditional Chinese Medicine to clinically treat bone diseases in China. Our previous studies showed that active ingredients of PT had a strong effect on enhancing proliferation and differentiation of Mesenchymal Stem Cells (MSCs) in vitro. What is more, PT was discovered to have a antiapoptosis effect to dopaminergic neuron of Parkinson rats. WhetherPT prompt proliferation of ESCs and against injury which cause ESCs to be apoptotic, together with its mechanism still be unknown. We hope to find a new way for clinical skin damage and exploring skin beauty products through our research.
Methods
(1) ESCs'cultivation and purification.
We used back skin of fatal rats which come from the two-weeks pregnant rats' womb, cut into pieces with scissors, collected cells for cultivation, stained cells from passageⅠtoⅣwith ESCs'special marker keratin19 (K19) andβ1-intergrin, counted double stained cells using fluorescence, flow cytometry (FCM), tested cells, property and purity, and choosen the most purest cells passageⅢto study.
(2) Active ingredients of PT inhibited ESCs' apoptosis caused by Ultra-Violet radiation.
We divided cells into three groups in light of different radiation time,5 minutes,15 minutes or 30 minutes, based on certain injury dosage of 100mJ/cm2, kept on culturing for 10 hours,20 hours or 36 hours. After that we tested these groups, apoptotic percentage by FCM, and confirmed that ESCs appear to ultraviolet 15 minutes and then culture 20 hours make a stable apoptosis model.
Divided cells into normal group, control group and drug groups. Drug groups included 2B group, S6 group (stearic acid ethyl ester), S8 group (tetradecanoic acid sterol ester), and S9 group((+)-4-Cholesten-3-one). No special treatment to the normal group and delt with the control group like the last paragraph. Differences between control group and drug groups are that drug groups added 2B, S6, S8, or S9 in the basic medium after lesion. Comparision of anti-apoptotic effects of different parts of the active ingredients of PT by flow cytometry testing ESCs' apoptotic rate. Immunohistochemical staining of p53, caspase3 and bcl-2 to test positive expression. Western blot method to detect protein expressions of socs7, nck, p53 and caspase3.
(3) Active ingredients of PT inhibited ESCs' apoptosis caused by serum-deprivated culture.
Planted cells into 96-well-plates. Disposed of the medium of control and drug groups' wells, and washed the wells with D-Hank's after 24h cultivation except the normal group. Added basic medium to the control group, and extra active ingredient of PT to the drug groups, cultured 24h, 48h, and 72h. tested these groups' optical density (OD) values by MTT method to make sure the best time of drug effect. Tested anti-apoptotic effect of the active ingredients of PT by flow cytometry. Western blot method to detect protein expressions of bcl-2 and caspase3.
Results
(1) ESCs'cultivation and purification.
The back skin of fetal rats contain aboundance of ESCs. Results of an examination tested by FCM revealed that the positive rate of ESCs double stained with K19 andβ1-intergrin had a peak value in passage III.
(2) Active ingredients of PT inhibited ESCs' apoptosis caused by Ultra-Violet radiation.
The normal rate, apoptosis rate and mortality rate of ESCs were more rational under condition that 20h cultivation after 15min radiation damage dosage of 100mJ/c m2,and applicable to be an apoptotic model induced by Ultra-Violet radiation. Immunohistochemistry results showed groups of PTE had more positive cells stained with bcl-2 than the control group and opposite outcomes with caspase.3 and p53. Results of western blot indicated that groups S8 and S9 had a obvious decrease in expression of socs7, nck, p53, and caspase3.
(3) Active ingredients of PT inhibited ESCs' apoptosis caused by serum-deprivated culture.
As MTT results showed PTE acting on ESCs which cultured in serum deprived medium had a peak value of OD in 48h; results of FCM revealed that drug groups had a better anti-apoptotic effect compared with control group in 48h; western blot outcomes indicated that all the drug groups up-regulated bcl-2 and down-regulated caspase3,2B had a outstanding performance.
Conclusion
(1) Back skin of fetal rat have aboundance of ESCs' and the passageⅢcells reach a biggest percentage of ESCs.
(2) Active ingredients of PT inhibit ESCs' apoptosis caused by Ultra-Violet radiation, S8 and S9 have obviously anti-apoptotic effect.
(3) Active ingredients of PT inhibited ESCs' apoptosis caused by serum-deprivated culture, and 2B has a notable activity.
干细胞是目前生命科学研究的重点和热点,其中表皮干细胞是解决各种皮肤问题的重要途径。表皮干细胞(epidermal stem cells, ESCs)具有易分离、易扩增及体外操作简便等特点,所以在临床上有较广泛的应用前景。紫外辐射和失营养是造成皮肤损伤的重要因素,因此,寻找安全有效的抗表皮干细胞紫外辐射凋亡、失营养凋亡的方法是当前研究的重要方向。
龟板具有滋阴潜阳,补肾壮骨,养血补心等功效。本研究课题组早前实验发现,龟板有效成分PTE(龟板乙酸乙酯部位提取物,简称2B)在早期可以促进骨髓间充质干细胞(mesenchvmal stem cells, MSCs)的增殖,继续培养,则促进了MSCs向成骨细胞的分化。另外还发现龟板对帕金森病大鼠多巴胺能神经元具有抗凋亡的作用。龟板作为一种补肾中药是否也可以促进ESCs增殖,对抗损伤的ESCs凋亡,及其相关作用机制是什么,目前还不清楚,因此本实验希望通过龟板有效成分对ESCs影响的研究,从而为临床医学皮肤损伤修复及护肤美容产品开发提供一种可能的新的途径。
二、研究方法
(1)表皮干细胞的培养与纯化
本实验取用孕两周Sprague-Dawley (SD)大鼠腹中胎鼠的背部皮肤,剪碎后加入胰酶消化,并收集细胞进行培养。收集第Ⅰ至Ⅳ代细胞,采用ESCs的标记物角蛋白19(keratin19, K19)和β1整合素(β1-integrin)进行双标鉴定,检测所培养细胞的性质和纯度,选取纯度最高的第Ⅲ代表皮干细胞进行以下实验。(2)龟板有效成分抗表皮干细胞紫外线损伤凋亡实验
确定紫外损伤模型：将细胞种入6孔板,培养24h后,吸出孔内的培养基,并用D-Hank's液清洗,按一定损伤量(100mJ/cm2)、不同损伤时间分组,分为三组,分别紫外照射5min、15min、30min, D-Hank's液清洗,加入培养基继续培养10h、20h、36h,之后进行流式细胞术AnnexinⅤ/PI双标检测各组凋亡率,依据凋亡率结果确定：紫外损伤15min,继续培养20h可获得稳定的凋亡模型。
药物干预：将细胞分为正常组、模型组、药物组。药物组又分为2B组、S6组(十八酸乙酯)、S8组(十四酸甾醇酯)、S9组(4-甾酮)。正常组无特殊处理,模型组方法同前,药物组与模型组不同的是,损伤后所加培养基中添加2B、S6、S8或S9,通过流式细胞术测定凋亡率来比较龟板有效成分不同部位抗凋亡的效果；免疫组化法观察各组中p53、caspase3和bcl-2的阳性表达情况；蛋白免疫印迹法检测socs7、nck、p53、caspase3等蛋白的表达水平。
(3)龟板有效成分抗表皮干细胞无血清损伤凋亡实验
将细胞种入96孔板,培养24h后,正常组不做特殊处理,吸出模型组、药物组孔内的培养基,并用D-Hank's液清洗,模型组加入无血清培养基,药物组加入含龟板有效成分的无血清培养基,分别培养24h、48h、72h,MTT法检测各组光密度值(OD值),确立最佳药效时段。通过流式细胞术检测最佳时段中各种药物组细胞凋亡率,比较各药物组抗凋亡的效果。免疫印迹检测caspase3和bcl-2的表达情况。
三、实验结果
(1)表皮干细胞的培养与纯化
流式细胞术检测第Ⅰ至Ⅳ代细胞K19和β1整合素双标阳性百分率平均值分别是89.75%、94.55%、96.57%、78.75%。结果提示：胎鼠背部皮肤中ESCs含量丰富,第Ⅲ代表皮干细胞K19和β1整合素的双标阳性率明显高于第Ⅰ、Ⅱ、Ⅳ代细胞,说明第Ⅲ代表皮干细胞纯度最高。
(2)龟板有效成分抗表皮干细胞紫外线损伤凋亡实验
表皮干细胞在100mJ/cm2紫外损伤量下照射15min,再培养20h,与其他损伤时间组(5min组、30min组)、其他损伤后培养时间组(12h组、36h组)相比,细胞正常率较高,凋亡率较高,死亡率较低,可作为紫外辐射凋亡模型。免疫组化结果显示：bcl-2在细胞浆和细胞核均有表达,caspase3表达于细胞浆中,P53主要表达于细胞核内。龟板有效成分各组中bcl-2阳性细胞数多于模型组,而caspase3、p53阳性细胞数少于模型组。免疫印迹结果显示PTE中S8、S9组明显降低socs7、nck、p53、caspase3等蛋白的表达水平。
(3)龟板有效成分抗表皮干细胞无血清损伤凋亡实验
MTT结果表明,PTE作用于无血清培养的表皮干细胞,48h时0D值高于24h组、72h组；流式检测结果表明48h时各药物组与模型组相比,有较好的抗凋亡作用；免疫印迹显示各药物组均上调bcl-2,下调caspase3,其中以2B效果最明显。
四、结论
(1)胎鼠背部皮肤ESCs含量丰富；细胞培养至第Ⅲ代, ESCs纯度最高。
(2)龟板有效成分抗表皮干细胞紫外线损伤凋亡,其中S8、S9抗凋亡效果最明显。
(3)龟板有效成分抗表皮干细胞无血清损伤凋亡,以2B效果最明显。
Objective
Stem cells is one of the most attracting fields in the life sciences in the recent decades and Epidermal Stem Cells (ESCs) is the significant approach to deal with various kinds of skin problems. ESCs is one of stem cells, which has the characteristic that it is easy to separate, proliferate and culture in vitro, has widespreadly applying prospect in clinic. Ultra-Violet (UV) radiation and nutriment deficiency are the significant causes to the skin damage, so it is the most important problem that how to find an safe and effective method to inhibit ESCs apoptosis induced by Ultra-Violet radiation and nutriment deficiency. Based on Traditional Chinese Medicine theory that "the kidney being the origin of congenital constitution, controlling growth and reproduction", we hope to promote ESCs proliferation. and resist apoptosis with the Chinese Medicine PlastrumTestudinis (PT) which can invigorate the kidney.
Traditional Chinese Medicine holds that PT has the efficacy of nourishing liver and kidney, so it is often used as an important Traditional Chinese Medicine to clinically treat bone diseases in China. Our previous studies showed that active ingredients of PT had a strong effect on enhancing proliferation and differentiation of Mesenchymal Stem Cells (MSCs) in vitro. What is more, PT was discovered to have a antiapoptosis effect to dopaminergic neuron of Parkinson rats. WhetherPT prompt proliferation of ESCs and against injury which cause ESCs to be apoptotic, together with its mechanism still be unknown. We hope to find a new way for clinical skin damage and exploring skin beauty products through our research.
Methods
(1) ESCs'cultivation and purification.
We used back skin of fatal rats which come from the two-weeks pregnant rats' womb, cut into pieces with scissors, collected cells for cultivation, stained cells from passageⅠtoⅣwith ESCs'special marker keratin19 (K19) andβ1-intergrin, counted double stained cells using fluorescence, flow cytometry (FCM), tested cells, property and purity, and choosen the most purest cells passageⅢto study.
(2) Active ingredients of PT inhibited ESCs' apoptosis caused by Ultra-Violet radiation.
We divided cells into three groups in light of different radiation time,5 minutes,15 minutes or 30 minutes, based on certain injury dosage of 100mJ/cm2, kept on culturing for 10 hours,20 hours or 36 hours. After that we tested these groups, apoptotic percentage by FCM, and confirmed that ESCs appear to ultraviolet 15 minutes and then culture 20 hours make a stable apoptosis model.
Divided cells into normal group, control group and drug groups. Drug groups included 2B group, S6 group (stearic acid ethyl ester), S8 group (tetradecanoic acid sterol ester), and S9 group((+)-4-Cholesten-3-one). No special treatment to the normal group and delt with the control group like the last paragraph. Differences between control group and drug groups are that drug groups added 2B, S6, S8, or S9 in the basic medium after lesion. Comparision of anti-apoptotic effects of different parts of the active ingredients of PT by flow cytometry testing ESCs' apoptotic rate. Immunohistochemical staining of p53, caspase3 and bcl-2 to test positive expression. Western blot method to detect protein expressions of socs7, nck, p53 and caspase3.
(3) Active ingredients of PT inhibited ESCs' apoptosis caused by serum-deprivated culture.
Planted cells into 96-well-plates. Disposed of the medium of control and drug groups' wells, and washed the wells with D-Hank's after 24h cultivation except the normal group. Added basic medium to the control group, and extra active ingredient of PT to the drug groups, cultured 24h, 48h, and 72h. tested these groups' optical density (OD) values by MTT method to make sure the best time of drug effect. Tested anti-apoptotic effect of the active ingredients of PT by flow cytometry. Western blot method to detect protein expressions of bcl-2 and caspase3.
Results
(1) ESCs'cultivation and purification.
The back skin of fetal rats contain aboundance of ESCs. Results of an examination tested by FCM revealed that the positive rate of ESCs double stained with K19 andβ1-intergrin had a peak value in passage III.
(2) Active ingredients of PT inhibited ESCs' apoptosis caused by Ultra-Violet radiation.
The normal rate, apoptosis rate and mortality rate of ESCs were more rational under condition that 20h cultivation after 15min radiation damage dosage of 100mJ/c m2,and applicable to be an apoptotic model induced by Ultra-Violet radiation. Immunohistochemistry results showed groups of PTE had more positive cells stained with bcl-2 than the control group and opposite outcomes with caspase.3 and p53. Results of western blot indicated that groups S8 and S9 had a obvious decrease in expression of socs7, nck, p53, and caspase3.
(3) Active ingredients of PT inhibited ESCs' apoptosis caused by serum-deprivated culture.
As MTT results showed PTE acting on ESCs which cultured in serum deprived medium had a peak value of OD in 48h; results of FCM revealed that drug groups had a better anti-apoptotic effect compared with control group in 48h; western blot outcomes indicated that all the drug groups up-regulated bcl-2 and down-regulated caspase3,2B had a outstanding performance.
Conclusion
(1) Back skin of fetal rat have aboundance of ESCs' and the passageⅢcells reach a biggest percentage of ESCs.
(2) Active ingredients of PT inhibit ESCs' apoptosis caused by Ultra-Violet radiation, S8 and S9 have obviously anti-apoptotic effect.
(3) Active ingredients of PT inhibited ESCs' apoptosis caused by serum-deprivated culture, and 2B has a notable activity.
引文
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