牛黄天龙饮含药血清诱导人宫颈癌HeLa细胞凋亡及其机理的研究
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摘要
目的：宫颈癌是最常见的女性生殖道肿瘤，它严重威胁着妇女的健康和生命。一般认为，浸润性宫颈癌的治疗是手术和放疗。由于手术的局限性及放疗易引起病灶旁正常脏器的永久性损伤和继发性肿瘤，使人们需要寻找一种新的治疗方案。近年来，有一种倾向就是把辅助化疗加同时放疗作为晚期宫颈癌的治疗标准，目的是使瘤体缩小，减少局部复发率及远处转移率。因此作为辅助化疗的药物就成为人们关注的热点。多种组合的化疗方案已被临床医务人员探讨、研究，但化学药物不可避免的毒副反应成为化疗中的亟待解决的又一难题。为寻找新的毒副作用小的化疗药物人们把目光投向了祖国医学，关于中药诱导肿瘤细胞凋亡的研究多有报导，但细胞凋亡是一个非常复杂的过程，受多种因素的调节和制约。根据这一原理，复方中药可能具有“多靶作用”的作用机理显示出了独特的优越性。本实验根据祖国传统医学知识，在传统组方“犀黄丸”的基础上组建了复方中药“牛黄天龙饮”，探讨其对HeLa细胞的凋亡作用及其机理,采用一系列研究凋亡的典型方法，为中药治疗肿瘤的研究提供了一种新思路、新方法。
方法：SD大鼠，50只，雌性，随机分为中药组（高、中、低剂量组）、空白对照组、阳性对照组即化疗药物组（DDP组），每组10只。中药组分别给以高、中、低三个剂量的中
药灌胃，空白对照组给以等容积生理盐水灌胃，化疗药物组给以等容积顺铂腹腔注射，3天后，无菌条件下取血，以2000转/分的转速，离心10分钟获取含药血清，经真空冻干制成血清冻干粉。HeLa细胞和二倍体细胞（人胚肺成纤维细胞）在含10%和20%的胎牛血清RPMI-1640培养基中，于37℃，CO2含量5.0%，湿度95%的孵箱中培养。以相同浓度接种于96孔板中，待细胞贴壁进入对数生长期后，换10%含药血清培养48小时后用噻唑兰（MTT）方法检测各试验组的含药血清对HeLa细胞增殖的抑制情况，以人胚肺成纤维细胞作对照，比较中药含药血清组与化疗药物组对二倍体细胞的抑制作用。用相同的方法处理细胞后收集细胞悬液，用吖啶橙/溴化乙锭（AO/EB）双荧光染色法观察细胞凋亡的形态学变化；用流式细胞术、DNA琼脂糖凝胶电泳、DNA缺口末端标记法（TUNEL）检测凋亡，并用流式细胞术检测经含药血清处理后Bcl-2， c-Myc基因蛋白在人宫颈癌HeLa细胞中的表达变化，对药物的作用机理进行初步探讨。
结果：1 MTT法：人宫颈癌HeLa细胞在10%的药物血清作用24、48小时后，增殖率受到抑制，且呈时间依赖性及浓度依赖性。高、中、低剂量组和化疗药物组24小时的细胞抑制率分别为5.26%，10.52%，5.26%，32.89%；48小时分别为2.38%，23.80%，13.10%，7.14%。在相同的时间内，中剂量组与低剂量组相比抑制率升高，有显著差异（P<0.05）。在不同的时间内，除高剂量组外同一试验组之间相比差异有显著性（P<0.01）。人胚肺成纤维细胞在10%的药物血清作用24、48小时后，可见化疗药物组细胞增殖受到抑制，而不同浓度的中药组含药血清对二倍体细胞的生
长有一定的促进作用。48小时高、中、低剂量组的细胞生存率分别为178.57%, 228.57%, 314.28%，与对照组相比有显著性差异（P<0.01）。2 AO/EB双荧光染色法：空白对照组以正常细胞为主（核被AO染色，呈黄绿色-黄色荧光，密度不均，呈现结构样特征），低剂量及高剂量中药组仍以正常细胞为主，早期凋亡细胞比空白对照组多。中剂量中药组和化疗药物组正常细胞减少，凋亡细胞增多，可见晚期凋亡细胞（核被EB染色呈橙红色，可见核的断裂和浓缩，并可见凋亡小体）及坏死细胞（核被EB染色呈橙红色，结构不清，细胞体积增大，边缘模糊）。3 流式细胞术：与空白对照组相比，不同剂量的中药组及化疗药物组细胞凋亡率明显升高（P<0.01）， G1期细胞增多（P<0.01）， S期细胞减少（P<0.05）。, Bcl-2基因蛋白的表达在中剂量组与空白对照组相比降低，有显著性差异（P<0.05）； c-Myc基因蛋白的表达在中剂量组与空白对照组相比升高，有极显著性差异（P<0.01）。4 DNA琼脂糖凝胶电泳：化疗药物组、中剂量组及低剂量组可见典型的DNA梯状带，对照组电泳DNA呈现连续性。5 DNA缺口末端标记法：对照组可见少量的TUNEL阳性细胞（核被染成棕褐色颗粒）。试验组可见大量阳性细胞。高、中、低剂量组及化疗药物组细胞阳性率分别为11.95%，29.50%，17.10%，37.30%。各组与空白对照组相比有显著性差异（P<0.01）。
结论：1 复方中药“牛黄天龙饮”含药血清能诱导人宫颈癌HeLa细胞凋亡。2 中药组的抑制率虽然低于化疗药物组较低，但其对二倍体细胞生长的影响明显不同于化疗药物组，各中药组含药血清对二倍体细胞的生长有明显促进作
用。3 复方中药“牛黄天龙饮”含药血清降低Bcl-2基因的表达同时升高c-Myc基因的表达，这可能是其诱导HeLa细胞凋亡的途径之一。
Objective：Cervical cancer is the most common gynecologic tumor of genital duct. It threat women’s health and life. In general, for invasive carcinoma of uterine cervix, the treatment is surgery and radiotherapy. Because of the limit of surgery and radiotherapy inducing persistent injury to normal organs around the focus and secondary tumor, a new treatment for advanced cervical cancer is needed. In recent years, there is a trend that neoadjuvant chemotherapy and concurrent radiotherapy are regarded as the standard treatment. The goal of the treatment is to reduce tumor bulk and decrease local and distant recurrences. Thus the drugs used as neoajuvant chemotherapy became a hot topic. Combined chemotherapy agents have been studied. The inevitable problem of chemotherapeutic agent’s side effect makes us look for new drugs from traditional Chinese medicine. Chinese herbal inducing apoptosis has been reported, but apoptosis is a complex process and limited by many factors. Based on this theory, herbal mixture’s mechanism of multi-target is superior to others. “Niu Huang Tian Long Yin” is based on traditional Chinese herbal prescription “Xi Huang Wan” combined with
Hirudo, Fructus and so on. To explore the effect and mechanism of “Niu Huang Tian Long Yin” on cervical cancer cell HeLa, serologic pharmacology and a series of typical methods were used. The experiment provided a new thought and method for the study of antitumor.
Methods: SD rats were given different dose Chinese herb by gavage. After 3 days, rat’s blood was collected asepticly and serum was collected by centrifugation. Medical serum was made into powder through vaccum-freezing process. HeLa cell and human embryo lung cell were cultured in RPMI-1640 contained 10% or 20% FBS in 37.5℃, 5% CO2 HeLa cell were planted on 96-well plate on the same density and were affected by different concentration of contained medical serum for 48 hours, then cell proliferation inhibition was measured by MTT. Cell apoptosis was measured by AO/EB double fluorescent dye staining, flow cytometry, DNA agarose gel electrophoresis and TUNEL. The expression of Bcl-2, c-Myc was measured by flow cytometry.
Results: 1 MTT: Cervical cancer cell line HeLa was effected by medical serum for 24 or 48 hours, then cell proliferation inhibition was observed and it was in dose-dependent manner, time-dependent manner (P<0.05). The inhibition rate of HeLa cell was 5.26%, 10.52%, 5.26% and 32.89% respectively in different groups treated for 24 hours. The inhibition rate was 2.38%, 23.80%, 13.10% and 7.14% respectively in different groups treated for 48 hours. The effect
of medical serum on human embryo lung cell was different with on HeLa cell. Medical serum of Chinese herbal could promote the proliferation of diploid cell. 2 AO/EB double fluorescent dye staining: There were lots of normal cells but a few of apoptotic cells in control group and high dose group. There were more apoptotic cells in middle dose group and chemotherapeutic agent group and necrosis cell could be observed. 3 Flow cytometry: Apoptotic rate was different between trial groups and control group (P<0.05). Cells in S phase were reduced and in G1 phase were enhanced in trial groups (P<0.05). The expression of Bcl-2 was decreased in middle dose group. On the contrary, c-Myc was incresed. 4 DNA agarose gel electrophoresis: DNA ladder could be found in trial groups. 5 TUNEL: A few of positive cells were observed in control group while a lot of positive cells in trial groups. The positive rate was 11.95%, 29.50%, 17.10%, 37.30% and 4.68% respectively in high dose group, middle dose group, low dose group, chemotherapeutic agent group and control group.
Conclusions: 1 Cancer cell line HeLa could be induced apoptosis by Herbal mixture “Niu Huang Tian Long Yin”. 2 Apoptosis rate of herbal groups is lower than chemotherapeutic agent group. But medical serum of herbal groups could promote the proliferation of diploid cell. 3 “Niu Huang Tian Long Yin” could reduce the expression of Bcl-2 and enhance t
方法：SD大鼠，50只，雌性，随机分为中药组（高、中、低剂量组）、空白对照组、阳性对照组即化疗药物组（DDP组），每组10只。中药组分别给以高、中、低三个剂量的中
药灌胃，空白对照组给以等容积生理盐水灌胃，化疗药物组给以等容积顺铂腹腔注射，3天后，无菌条件下取血，以2000转/分的转速，离心10分钟获取含药血清，经真空冻干制成血清冻干粉。HeLa细胞和二倍体细胞（人胚肺成纤维细胞）在含10%和20%的胎牛血清RPMI-1640培养基中，于37℃，CO2含量5.0%，湿度95%的孵箱中培养。以相同浓度接种于96孔板中，待细胞贴壁进入对数生长期后，换10%含药血清培养48小时后用噻唑兰（MTT）方法检测各试验组的含药血清对HeLa细胞增殖的抑制情况，以人胚肺成纤维细胞作对照，比较中药含药血清组与化疗药物组对二倍体细胞的抑制作用。用相同的方法处理细胞后收集细胞悬液，用吖啶橙/溴化乙锭（AO/EB）双荧光染色法观察细胞凋亡的形态学变化；用流式细胞术、DNA琼脂糖凝胶电泳、DNA缺口末端标记法（TUNEL）检测凋亡，并用流式细胞术检测经含药血清处理后Bcl-2， c-Myc基因蛋白在人宫颈癌HeLa细胞中的表达变化，对药物的作用机理进行初步探讨。
结果：1 MTT法：人宫颈癌HeLa细胞在10%的药物血清作用24、48小时后，增殖率受到抑制，且呈时间依赖性及浓度依赖性。高、中、低剂量组和化疗药物组24小时的细胞抑制率分别为5.26%，10.52%，5.26%，32.89%；48小时分别为2.38%，23.80%，13.10%，7.14%。在相同的时间内，中剂量组与低剂量组相比抑制率升高，有显著差异（P<0.05）。在不同的时间内，除高剂量组外同一试验组之间相比差异有显著性（P<0.01）。人胚肺成纤维细胞在10%的药物血清作用24、48小时后，可见化疗药物组细胞增殖受到抑制，而不同浓度的中药组含药血清对二倍体细胞的生
长有一定的促进作用。48小时高、中、低剂量组的细胞生存率分别为178.57%, 228.57%, 314.28%，与对照组相比有显著性差异（P<0.01）。2 AO/EB双荧光染色法：空白对照组以正常细胞为主（核被AO染色，呈黄绿色-黄色荧光，密度不均，呈现结构样特征），低剂量及高剂量中药组仍以正常细胞为主，早期凋亡细胞比空白对照组多。中剂量中药组和化疗药物组正常细胞减少，凋亡细胞增多，可见晚期凋亡细胞（核被EB染色呈橙红色，可见核的断裂和浓缩，并可见凋亡小体）及坏死细胞（核被EB染色呈橙红色，结构不清，细胞体积增大，边缘模糊）。3 流式细胞术：与空白对照组相比，不同剂量的中药组及化疗药物组细胞凋亡率明显升高（P<0.01）， G1期细胞增多（P<0.01）， S期细胞减少（P<0.05）。, Bcl-2基因蛋白的表达在中剂量组与空白对照组相比降低，有显著性差异（P<0.05）； c-Myc基因蛋白的表达在中剂量组与空白对照组相比升高，有极显著性差异（P<0.01）。4 DNA琼脂糖凝胶电泳：化疗药物组、中剂量组及低剂量组可见典型的DNA梯状带，对照组电泳DNA呈现连续性。5 DNA缺口末端标记法：对照组可见少量的TUNEL阳性细胞（核被染成棕褐色颗粒）。试验组可见大量阳性细胞。高、中、低剂量组及化疗药物组细胞阳性率分别为11.95%，29.50%，17.10%，37.30%。各组与空白对照组相比有显著性差异（P<0.01）。
结论：1 复方中药“牛黄天龙饮”含药血清能诱导人宫颈癌HeLa细胞凋亡。2 中药组的抑制率虽然低于化疗药物组较低，但其对二倍体细胞生长的影响明显不同于化疗药物组，各中药组含药血清对二倍体细胞的生长有明显促进作
用。3 复方中药“牛黄天龙饮”含药血清降低Bcl-2基因的表达同时升高c-Myc基因的表达，这可能是其诱导HeLa细胞凋亡的途径之一。
Objective：Cervical cancer is the most common gynecologic tumor of genital duct. It threat women’s health and life. In general, for invasive carcinoma of uterine cervix, the treatment is surgery and radiotherapy. Because of the limit of surgery and radiotherapy inducing persistent injury to normal organs around the focus and secondary tumor, a new treatment for advanced cervical cancer is needed. In recent years, there is a trend that neoadjuvant chemotherapy and concurrent radiotherapy are regarded as the standard treatment. The goal of the treatment is to reduce tumor bulk and decrease local and distant recurrences. Thus the drugs used as neoajuvant chemotherapy became a hot topic. Combined chemotherapy agents have been studied. The inevitable problem of chemotherapeutic agent’s side effect makes us look for new drugs from traditional Chinese medicine. Chinese herbal inducing apoptosis has been reported, but apoptosis is a complex process and limited by many factors. Based on this theory, herbal mixture’s mechanism of multi-target is superior to others. “Niu Huang Tian Long Yin” is based on traditional Chinese herbal prescription “Xi Huang Wan” combined with
Hirudo, Fructus and so on. To explore the effect and mechanism of “Niu Huang Tian Long Yin” on cervical cancer cell HeLa, serologic pharmacology and a series of typical methods were used. The experiment provided a new thought and method for the study of antitumor.
Methods: SD rats were given different dose Chinese herb by gavage. After 3 days, rat’s blood was collected asepticly and serum was collected by centrifugation. Medical serum was made into powder through vaccum-freezing process. HeLa cell and human embryo lung cell were cultured in RPMI-1640 contained 10% or 20% FBS in 37.5℃, 5% CO2 HeLa cell were planted on 96-well plate on the same density and were affected by different concentration of contained medical serum for 48 hours, then cell proliferation inhibition was measured by MTT. Cell apoptosis was measured by AO/EB double fluorescent dye staining, flow cytometry, DNA agarose gel electrophoresis and TUNEL. The expression of Bcl-2, c-Myc was measured by flow cytometry.
Results: 1 MTT: Cervical cancer cell line HeLa was effected by medical serum for 24 or 48 hours, then cell proliferation inhibition was observed and it was in dose-dependent manner, time-dependent manner (P<0.05). The inhibition rate of HeLa cell was 5.26%, 10.52%, 5.26% and 32.89% respectively in different groups treated for 24 hours. The inhibition rate was 2.38%, 23.80%, 13.10% and 7.14% respectively in different groups treated for 48 hours. The effect
of medical serum on human embryo lung cell was different with on HeLa cell. Medical serum of Chinese herbal could promote the proliferation of diploid cell. 2 AO/EB double fluorescent dye staining: There were lots of normal cells but a few of apoptotic cells in control group and high dose group. There were more apoptotic cells in middle dose group and chemotherapeutic agent group and necrosis cell could be observed. 3 Flow cytometry: Apoptotic rate was different between trial groups and control group (P<0.05). Cells in S phase were reduced and in G1 phase were enhanced in trial groups (P<0.05). The expression of Bcl-2 was decreased in middle dose group. On the contrary, c-Myc was incresed. 4 DNA agarose gel electrophoresis: DNA ladder could be found in trial groups. 5 TUNEL: A few of positive cells were observed in control group while a lot of positive cells in trial groups. The positive rate was 11.95%, 29.50%, 17.10%, 37.30% and 4.68% respectively in high dose group, middle dose group, low dose group, chemotherapeutic agent group and control group.
Conclusions: 1 Cancer cell line HeLa could be induced apoptosis by Herbal mixture “Niu Huang Tian Long Yin”. 2 Apoptosis rate of herbal groups is lower than chemotherapeutic agent group. But medical serum of herbal groups could promote the proliferation of diploid cell. 3 “Niu Huang Tian Long Yin” could reduce the expression of Bcl-2 and enhance t
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