鼻咽癌细胞株CNE-2Z中肿瘤干细胞样细胞的生物学特性及益气解毒方干预效应研究
发布时间：2018-08-14 19:52
【摘要】：第一章：鼻咽癌细胞株CNE-2Z中肿瘤干细胞样细胞的悬浮培养和鉴定目的：探讨富集鼻咽癌肿瘤干细胞样细胞的方法并鉴定鼻咽癌细胞株CNE-2Z中的肿瘤干细胞样细胞。方法：(1)取鼻咽癌细胞株CNE-2Z普通培养至对数生长期的细胞,消化后分别细胞计数,密度达到1×103/ml.1×104/m1、1×105/ml、1×106 ／ml时,分别加入无血清培养液中继续培养,以0.3%台盼蓝计数第0、1、3、5、7、9天活细胞百分率；(2)制备普通培养活细胞和第9天无血清培养活细胞爬片,用CD133和CD44人抗鼠单克隆抗体免疫组化染色；(3)用第9天无血清培养活细胞制备电镜观察标本并进行电镜观察。结果：(1)细胞密度为1×103／ml.1×10/ml.1×105/ml.1×106/ml时的细胞悬液再经无血清悬浮培养至第9天时,其中的活细胞百分率分别是0.00%、0.05%、3.10%、0.27%,以1 x1 05/ml组活细胞比率最高；(2)普通培养细胞爬片的CD133和CD44细胞强阳性率分别是2.71%和3.00%,而无血清培养细胞爬片的CD133和CD44强阳性率分别为66.7%和67.00%,两种培养方法强阳性细胞比较,差异有非常显著性统计学意义(P0.01),CD133和CD44在两种培养液中强阳性细胞百分率相关性分析呈正相关(P0.01)；(3)扫描电镜和透射电镜观察所见CNE-2Z鼻咽癌细胞中肿瘤干细胞样细胞表面有鳞片样改变,伴有出芽样突起,透射电镜下,细胞呈现较大异质性,可为圆形或椭圆形,细胞核固缩,细胞质极度浓缩和细胞器严重减少,可见类凋亡(?)小体以类似出胞的方式排出胞外。结论：1.无血清悬浮培养法可富集CNE-2Z鼻咽癌细胞中的肿瘤干细胞样细胞,1×105/ml的细胞浓度是CNE-2Z鼻咽癌细胞的最佳悬浮培养细胞密度。无血清悬浮培养法是一种比较经济、易于掌握的培养肿瘤干细胞的较好方法,值得推广应用。2.CD1 33和CD44可作为鼻咽癌肿瘤干细胞样细胞的表面标志物而应用于鼻咽癌肿瘤干细胞样细胞的检测；在无血清悬浮培养液中,鼻咽癌肿瘤干细胞样细胞以自我复制和增殖两种方式分裂生长,但生长缓慢。3.电子显微镜下,鼻咽癌肿瘤干细胞样细胞的形态特征有较大异质性,细胞核固缩,细胞质极度浓缩和细胞器严重减少,细胞质中可见类凋亡(?)小体以类似出胞的方式排出胞外,这些变化可能与无血清环境改变了细胞的生物学特性有关。第二章：鼻咽癌细胞株CNE-2Z肿瘤干细胞样细胞的生物学特性及益气解毒方对其分化潜能的干预效应目的：研究鼻咽癌细胞株CNE-2Z中肿瘤干细胞样细胞的生物学特性及益气解毒方对该类细胞分化潜能的干预效应。方法：(1)采用无血清悬浮培养方法富集CNE-2Z鼻咽癌细胞中的肿瘤干细胞样细胞球；(2)分别采用无血清悬浮培养转含胎牛血清的普通培养法及持续无血清培养法检测培养体系中悬浮细胞球的克隆形成能力、分化增殖能力,并以不同密度的细胞球悬液注射至裸鼠皮下,观察其成瘤能力；(3)用益气解毒方含药血清配制的温育液与无血清悬浮培养所得肿瘤干细胞样细胞球共培养,分别设立普通组和空白对照组,比较24 hrs、72 hrs、5天等时间段时细胞球中CD133/CD44阳性细胞数的变化。结果：(1)将培养体系中获得的CNE-2Z鼻咽癌细胞球吹散后再行无血清悬浮培养,仍然可形成细胞球；将这样的细胞球转至普通培养,24 hrs后可见在培养体系中有单个细胞贴壁生长,7天后细胞球消失；贴壁生的细胞的形态特征与原亲代细胞无异；对这些贴壁细胞的免疫组化检测显示CD133+/CD44+细胞阳性率逐日下降,各时段比较均有统计学意义(P0.05)；交替培养时,目标细胞在细胞球和贴壁细胞之间交替出现,细胞(球)形态无明显改变；(2)无血清悬浮培养细胞球经洗涤后,分别以1×103、1×105、1×107的细胞密度进行裸鼠皮下注射,均可形成移植瘤,肿瘤生长速度与注射的细胞密度有关；移植瘤原代培养细胞生长速度明显快于其来源的亲代细胞株细胞(P0.05)；(3)益气解毒方含药血清温育液中CD133/CD44阳性细胞下降速率明显快于普通培养组和对照组,各时段与普通组、对照组比较均有统计学意义(P0.05)。结论：1、无血清悬浮培养法所得CNE-2Z鼻咽癌细胞球具有自我更新、高速分化增殖和高效体内成瘤等肿瘤干细胞特性,可能为其中的肿瘤干细胞样细胞群。2、益气解毒方体外作用对CNE-2Z鼻咽癌细胞中的肿瘤干细胞样细胞具有明显的分化诱导效应。第三章：益气解毒方对CNE-2Z鼻咽癌细胞裸鼠移植瘤中肿瘤干细胞样细胞干预作用的初步研究目的：探讨益气解毒方对CNE-2Z鼻咽癌细胞裸鼠移植瘤组织中肿瘤干细胞样细胞的干预效应,比较不同给药方案对这类肿瘤干细胞样细胞的干预效率以及对裸鼠生存质量的影响。方法：建立CNE-2Z鼻咽癌细胞裸鼠移植瘤模型；随机分为模型组、中药组(益气解毒方组)、DDP组(顺铂)和联合用药组(益气解毒方+DDP),分别给予生理盐水、益气解毒方药液、顺铂单药和益气解毒方药液+顺铂处理；观察用药前后各组移植瘤裸鼠体重、皮肤颜色、移植瘤瘤体大小、一般生活状态等相关指标变化；用药2周后获取移植瘤瘤组织标本,免疫组织化学染色法测定并分析比较各组移植瘤组织细胞CD133和CD44表达情况,western blotting法检测各组移植瘤组织CD133和CD44蛋白含量。结果：1)四组移植瘤裸鼠用药前后的体重变化表现为：模型组和益气解毒方组用药前后体重比较有非常显著性差异(P0.01),联合用药组和DDP组用药前后体重比较无显著性差异(P0.05)；移植瘤瘤体大小变化表现为：益气解毒方组、联合用药组、DDP组三组与模型组比较均有显著性差异(P0.05),益气解毒方组、联合用药组、DDP组抑瘤率依次为18.7%、58.4%、43.8%；皮肤颜色和生活状态变化表现为：模型组和益气解毒方组用药前后无明显差异,联合用药组和DDP组用药前后有显著性差异,即皮肤颜色变暗,生活活力下降,尤其DDP组明显。2)四组裸鼠移植瘤中CD133/CD44蛋白质的western blotting检测结果为：在Mark条带120 kDa.80-95 kDa分子量附近均出现相应蛋白质条带。但各组移植瘤标本的相应蛋白质条带亮度有差异。模型组最亮,益气解毒方组和DDP组较亮,联合用药组最暗。联合用药组与DDP组、益气解毒方组、模型组比较亮度差异有显著性。3)四组裸鼠移植瘤组织中CD133和CD44免疫组化检测结果：CD133的表达为各用药组与模型组比较均有显著性差异(P0.05),益气解毒方组、DDP组与联合用药组比较有显著性差异(P0.05),益气解毒方组与DDP组比较无显著性差异(P0.05)；CD44的表达结果与CD133相近,即各用药组与模型组比较均有显著性差异(P0.05)；益气解毒方组、DDP组与联合用药组比较有显著性差异(P0.05)；益气解毒方组与DDP组比较无显著性差异(P0.05)；CD133和CD44比较,除联合用药组外,各组CD44阳性表达率明显高于CD133,二者相关性分析表现为高度正相关(P0.01)。结论1.CNE-2Z鼻咽癌细胞移植瘤组织中存在少量鼻咽癌肿瘤干细胞样细胞,且CD44阳性细胞表达率高于CD133；作为鼻咽癌肿瘤干细胞样细胞的表面标志物而言,二项指标中究竟哪一项具有更高的特异性,有待进一步阐明。2、顺铂单药组中CD133/CD44阳性细胞表达率明显高于联合用药组,提示抗癌药物顺铂可能有富集鼻咽癌肿瘤干细胞样细胞的作用；而中药益气解毒方可能对鼻咽癌肿瘤干细胞样细胞具有诱导分化作用。3、在几种给药方案中,单纯使用中药的抑瘤能力有限,单纯使用化疗药则对荷瘤机体损害较大,中西医结合用药方案则在提高宿主生存质量、有效杀灭癌细胞乃至肿瘤干细胞方面具有协同作用,不啻为一种更为合理的用药选择。
[Abstract]:Chapter 1: Suspension culture and identification of tumor stem cell-like cells in nasopharyngeal carcinoma cell line CNE-2Z Objective: To explore the method of enriching tumor stem cell-like cells in nasopharyngeal carcinoma cell line CNE-2Z and identify tumor stem cell-like cells in nasopharyngeal carcinoma cell line CNE-2Z. When the cell density reached 1 103/ml.1 65 Results: (1) When the cell density was 1 x 103/ml.1 x 10/ml.1 x 105/ml.1 x 106/ml, the percentage of viable cells was 0.00%, 0.05%, 3.10%, 0.27%, respectively, when the cell suspension was cultured in serum-free medium for 9 days. (2) The strong positive rates of CD133 and CD44 cells were 2.71% and 3.00% respectively in common culture slices, 66.7% and 67.00% in serum-free culture slices, respectively. The difference between the two culture methods was statistically significant (P 0.01). CD133 and CD44 were in two groups. There was a positive correlation between the percentage of strong positive cells in the culture medium (P 0.01); (3) Scanning electron microscopy and transmission electron microscopy (TEM) showed that the surface of tumor stem cell-like cells in CNE-2Z nasopharyngeal carcinoma cells showed squamous-like changes, accompanied by budding protuberances. Under transmission electron microscopy, the cells showed large heterogeneity, which could be round or oval, and the nucleus of the cells was pyknosis. Conclusion: 1. Serum-free suspension culture can enrich tumor stem cell-like cells in CNE-2Z nasopharyngeal carcinoma cells, and the best suspension culture cell density is 1 X105 / ml. CD1 33 and CD44 can be used as the surface markers of nasopharyngeal carcinoma tumor stem cell-like cells for detection of nasopharyngeal carcinoma tumor stem cell-like cells; in serum-free suspension medium, nasopharyngeal carcinoma tumor stem cell-like cells are fine. Cells divide and grow in two ways of self-replication and proliferation, but grow slowly. Chapter 2: Biological characteristics of nasopharyngeal carcinoma cell line CNE-2Z and the intervention effect of Yiqi Jiedu Recipe on its differentiation potential Objective: To study the biological characteristics of tumor stem cell-like cells in nasopharyngeal carcinoma cell line CNE-2Z and the effects of Yiqi Jiedu Recipe on its differentiation potential. Methods: (1) Tumor stem cell-like cell spheres in CNE-2Z nasopharyngeal carcinoma cells were enriched by serum-free suspension culture method; (2) The cloning of suspension cell spheres was detected by serum-free suspension culture and continuous serum-free culture respectively. The ability of formation, differentiation and proliferation were observed by injecting different density of cell suspension into nude mice subcutaneously. (3) The tumor-forming ability was observed by co-culture of tumor stem cell-like cell spheres obtained from serum containing Yiqi Jiedu recipe and serum-free suspension culture. Results: (1) The CNE-2Z nasopharyngeal carcinoma cell spheres obtained in the culture system were blown away and then cultured in serum-free suspension, and the spheres could still form cell spheres. When the spheres were transferred to normal culture, a single cell adhered to the wall and grew fine after 7 days. The positive rate of CD133 + / CD44 + cells decreased day by day, and there was statistical significance (P 0.05). In the alternate culture, the target cells appeared alternately between the cell sphere and adherent cells, and the cells (spheres) were shaped. (2) Subcutaneous injection of serum-free suspension cultured cell spheres at the cell densities of 1 x 103, 1 x 105, and 1 x 107 after washing resulted in the formation of transplanted tumors, the growth rate of which was related to the density of injected cells, and the growth rate of primary cultured cells was significantly faster than that of parental cells (P 0.05). (3) The decrease rate of CD133/CD44 positive cells in serum culture medium containing Yiqi Jiedu Recipe was significantly faster than that in normal culture group and control group, and there was statistical significance (P 0.05) in comparison with normal group and control group. Tumor stem cell characteristics such as in vivo tumorigenesis, may be one of the tumor stem cell-like cells. 2, Yiqi Jiedu Recipe has obvious differentiation induction effect on CNE-2Z nasopharyngeal carcinoma cells in vitro. Chapter 3: Yiqi Jiedu Recipe on CNE-2Z nasopharyngeal carcinoma cells transplanted in nude mice tumor stem cell-like cells intervention Objective: To investigate the intervention effect of Yiqi Jiedu Recipe on tumor stem cell-like cells in CNE-2Z nasopharyngeal carcinoma xenografts in nude mice, and compare the intervention efficiency of different dosage regimens on these tumor stem cell-like cells and the quality of life in nude mice. The model was randomly divided into model group, Chinese medicine group (Yiqi Jiedu Fang group), DDP group (cisplatin) and combined treatment group (Yiqi Jiedu Fang + DDP), respectively, given saline, Yiqi Jiedu Fang liquid, cisplatin single drug and Yiqi Jiedu Fang liquid + cisplatin treatment; observed the weight of transplanted tumor, skin color, tumor size, 1. The expression of CD133 and CD44 in transplanted tumor tissues was detected by immunohistochemical staining, and the contents of CD133 and CD44 protein in transplanted tumor tissues were detected by Western blotting. Results: 1) The four groups of transplanted tumor tissues were detected before and after treatment. Weight changes showed: the model group and Yiqi Jiedu Fang before and after the treatment of body weight compared with a very significant difference (P 0.01), combined treatment group and DDP group before and after the weight of no significant difference (P 0.05); transplanted tumor size changes showed: Yiqi Jiedu Fang group, combined treatment group, DDP group compared with the model group were three groups. Significant difference (P 0.05), Yiqi Jiedu Prescription group, combined drug group, DDP group tumor inhibition rate was 18.7%, 58.4%, 43.8%; skin color and life state changes showed: model group and Yiqi Jiedu Prescription group before and after treatment, no significant difference, combined drug group and DDP group before and after treatment, that is, skin color darkening, life vitality. The Western blotting results of CD133/CD44 protein in four groups of nude mice transplanted tumor showed that there were corresponding protein bands near the molecular weight of Mark band 120 kDa.80-95 kDa. CD13 3 and CD44 immunohistochemical results in the transplanted tumor tissues of the four groups: the expression of CD13 3 in each group was significantly different from that in the model group (P 0.05). The expression of CD13 3 was significantly different between the treatment group and the model group (P 0.05). There was significant difference (P 0.05), there was no significant difference between Yiqi Jiedu Prescription group and DDP group (P 0.05); the expression of CD44 was similar to that of CD133, that is, there was significant difference between each drug group and model group (P 0.05); Yiqi Jiedu Prescription group, DDP group and combined drug group had significant difference (P 0.05); Yiqi Jiedu Prescription group and DDP group had no significant difference (P 0.05); Compared with CD133 and CD44, the positive expression rate of CD44 in each group was significantly higher than that of CD133, and the correlation analysis showed a high positive correlation (P 0.01). As a surface marker of nasopharyngeal carcinoma stem cell-like cells, which of the two indicators has higher specificity remains to be further clarified. 2. The expression rate of CD133/CD44 positive cells in cisplatin monotherapy group is significantly higher than that in combination therapy group, suggesting that cisplatin may enrich nasopharyngeal carcinoma stem cell-like cells. Yiqi Jiedu Decoction may induce differentiation of nasopharyngeal carcinoma stem cell-like cells. 3. In several drug regimens, the inhibition ability of traditional Chinese medicine alone is limited, while chemotherapy alone is harmful to the tumor-bearing organism. The combination of traditional Chinese and Western medicine regimen can improve the quality of life of the host and kill cancer cells effectively. The synergistic effect on cancer stem cells is a more rational choice.
【学位授予单位】：湖南中医药大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.63
本文编号：2183947
[Abstract]:Chapter 1: Suspension culture and identification of tumor stem cell-like cells in nasopharyngeal carcinoma cell line CNE-2Z Objective: To explore the method of enriching tumor stem cell-like cells in nasopharyngeal carcinoma cell line CNE-2Z and identify tumor stem cell-like cells in nasopharyngeal carcinoma cell line CNE-2Z. When the cell density reached 1 103/ml.1 65 Results: (1) When the cell density was 1 x 103/ml.1 x 10/ml.1 x 105/ml.1 x 106/ml, the percentage of viable cells was 0.00%, 0.05%, 3.10%, 0.27%, respectively, when the cell suspension was cultured in serum-free medium for 9 days. (2) The strong positive rates of CD133 and CD44 cells were 2.71% and 3.00% respectively in common culture slices, 66.7% and 67.00% in serum-free culture slices, respectively. The difference between the two culture methods was statistically significant (P 0.01). CD133 and CD44 were in two groups. There was a positive correlation between the percentage of strong positive cells in the culture medium (P 0.01); (3) Scanning electron microscopy and transmission electron microscopy (TEM) showed that the surface of tumor stem cell-like cells in CNE-2Z nasopharyngeal carcinoma cells showed squamous-like changes, accompanied by budding protuberances. Under transmission electron microscopy, the cells showed large heterogeneity, which could be round or oval, and the nucleus of the cells was pyknosis. Conclusion: 1. Serum-free suspension culture can enrich tumor stem cell-like cells in CNE-2Z nasopharyngeal carcinoma cells, and the best suspension culture cell density is 1 X105 / ml. CD1 33 and CD44 can be used as the surface markers of nasopharyngeal carcinoma tumor stem cell-like cells for detection of nasopharyngeal carcinoma tumor stem cell-like cells; in serum-free suspension medium, nasopharyngeal carcinoma tumor stem cell-like cells are fine. Cells divide and grow in two ways of self-replication and proliferation, but grow slowly. Chapter 2: Biological characteristics of nasopharyngeal carcinoma cell line CNE-2Z and the intervention effect of Yiqi Jiedu Recipe on its differentiation potential Objective: To study the biological characteristics of tumor stem cell-like cells in nasopharyngeal carcinoma cell line CNE-2Z and the effects of Yiqi Jiedu Recipe on its differentiation potential. Methods: (1) Tumor stem cell-like cell spheres in CNE-2Z nasopharyngeal carcinoma cells were enriched by serum-free suspension culture method; (2) The cloning of suspension cell spheres was detected by serum-free suspension culture and continuous serum-free culture respectively. The ability of formation, differentiation and proliferation were observed by injecting different density of cell suspension into nude mice subcutaneously. (3) The tumor-forming ability was observed by co-culture of tumor stem cell-like cell spheres obtained from serum containing Yiqi Jiedu recipe and serum-free suspension culture. Results: (1) The CNE-2Z nasopharyngeal carcinoma cell spheres obtained in the culture system were blown away and then cultured in serum-free suspension, and the spheres could still form cell spheres. When the spheres were transferred to normal culture, a single cell adhered to the wall and grew fine after 7 days. The positive rate of CD133 + / CD44 + cells decreased day by day, and there was statistical significance (P 0.05). In the alternate culture, the target cells appeared alternately between the cell sphere and adherent cells, and the cells (spheres) were shaped. (2) Subcutaneous injection of serum-free suspension cultured cell spheres at the cell densities of 1 x 103, 1 x 105, and 1 x 107 after washing resulted in the formation of transplanted tumors, the growth rate of which was related to the density of injected cells, and the growth rate of primary cultured cells was significantly faster than that of parental cells (P 0.05). (3) The decrease rate of CD133/CD44 positive cells in serum culture medium containing Yiqi Jiedu Recipe was significantly faster than that in normal culture group and control group, and there was statistical significance (P 0.05) in comparison with normal group and control group. Tumor stem cell characteristics such as in vivo tumorigenesis, may be one of the tumor stem cell-like cells. 2, Yiqi Jiedu Recipe has obvious differentiation induction effect on CNE-2Z nasopharyngeal carcinoma cells in vitro. Chapter 3: Yiqi Jiedu Recipe on CNE-2Z nasopharyngeal carcinoma cells transplanted in nude mice tumor stem cell-like cells intervention Objective: To investigate the intervention effect of Yiqi Jiedu Recipe on tumor stem cell-like cells in CNE-2Z nasopharyngeal carcinoma xenografts in nude mice, and compare the intervention efficiency of different dosage regimens on these tumor stem cell-like cells and the quality of life in nude mice. The model was randomly divided into model group, Chinese medicine group (Yiqi Jiedu Fang group), DDP group (cisplatin) and combined treatment group (Yiqi Jiedu Fang + DDP), respectively, given saline, Yiqi Jiedu Fang liquid, cisplatin single drug and Yiqi Jiedu Fang liquid + cisplatin treatment; observed the weight of transplanted tumor, skin color, tumor size, 1. The expression of CD133 and CD44 in transplanted tumor tissues was detected by immunohistochemical staining, and the contents of CD133 and CD44 protein in transplanted tumor tissues were detected by Western blotting. Results: 1) The four groups of transplanted tumor tissues were detected before and after treatment. Weight changes showed: the model group and Yiqi Jiedu Fang before and after the treatment of body weight compared with a very significant difference (P 0.01), combined treatment group and DDP group before and after the weight of no significant difference (P 0.05); transplanted tumor size changes showed: Yiqi Jiedu Fang group, combined treatment group, DDP group compared with the model group were three groups. Significant difference (P 0.05), Yiqi Jiedu Prescription group, combined drug group, DDP group tumor inhibition rate was 18.7%, 58.4%, 43.8%; skin color and life state changes showed: model group and Yiqi Jiedu Prescription group before and after treatment, no significant difference, combined drug group and DDP group before and after treatment, that is, skin color darkening, life vitality. The Western blotting results of CD133/CD44 protein in four groups of nude mice transplanted tumor showed that there were corresponding protein bands near the molecular weight of Mark band 120 kDa.80-95 kDa. CD13 3 and CD44 immunohistochemical results in the transplanted tumor tissues of the four groups: the expression of CD13 3 in each group was significantly different from that in the model group (P 0.05). The expression of CD13 3 was significantly different between the treatment group and the model group (P 0.05). There was significant difference (P 0.05), there was no significant difference between Yiqi Jiedu Prescription group and DDP group (P 0.05); the expression of CD44 was similar to that of CD133, that is, there was significant difference between each drug group and model group (P 0.05); Yiqi Jiedu Prescription group, DDP group and combined drug group had significant difference (P 0.05); Yiqi Jiedu Prescription group and DDP group had no significant difference (P 0.05); Compared with CD133 and CD44, the positive expression rate of CD44 in each group was significantly higher than that of CD133, and the correlation analysis showed a high positive correlation (P 0.01). As a surface marker of nasopharyngeal carcinoma stem cell-like cells, which of the two indicators has higher specificity remains to be further clarified. 2. The expression rate of CD133/CD44 positive cells in cisplatin monotherapy group is significantly higher than that in combination therapy group, suggesting that cisplatin may enrich nasopharyngeal carcinoma stem cell-like cells. Yiqi Jiedu Decoction may induce differentiation of nasopharyngeal carcinoma stem cell-like cells. 3. In several drug regimens, the inhibition ability of traditional Chinese medicine alone is limited, while chemotherapy alone is harmful to the tumor-bearing organism. The combination of traditional Chinese and Western medicine regimen can improve the quality of life of the host and kill cancer cells effectively. The synergistic effect on cancer stem cells is a more rational choice.
【学位授予单位】：湖南中医药大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.63
【参考文献】
相关期刊论文 前1条
1 崔雪梅;;化疗对妇科恶性肿瘤患者生存质量的影响[J];中国医药导报;2011年05期
相关硕士学位论文 前1条
1 高霞;中药苏木和新合成的化学药对血源性细胞的诱导作用研究[D];上海师范大学;2008年
，本文编号：2183947
本文链接：https://www.wllwen.com/yixuelunwen/wuguanyixuelunwen/2183947.html
最近更新
教材专著
