食管癌四种证候相关自身抗原表达与清热化痰方对其调节作用研究

发布时间：2018-06-12 23:36

本文选题：食管癌 + 自身抗原　；参考：《河南中医药大学》2016年硕士论文

【摘要】：目的:本研究分析食管癌相关自身抗原CK13、CK16、CaD、ACTG2在食管癌四种证候中的表达分布规律,进而探讨清热化痰方及其清热和化痰两个拆方对食管癌细胞增殖及其相关自身抗原表达的调节作用,为食管癌证候的分子病机和临床用药提供依据。方法:1.用食管癌自身抗原抗体和病人自身血清对食管癌组织进行western blot分析比较;2.运用免疫组化法分析CK13、CK16、CaD、ACTG2在四种证候食管癌正常组织、癌旁组织和癌组织中的表达;3.运用MTT法检测清热化痰方及其清热和化痰两个拆方对食管癌细胞EC109、EC9706和TE-1细胞增殖活性的影响;4.运用流式细胞术分析清热化痰方及其清热和化痰两个拆方对食管癌细胞EC109、EC9706和TE-1细胞周期的影响;5.运用western blot分析清热化痰方及其清热和化痰两个拆方对食管癌细胞EC109、EC9706和TE-1 CK13、CK16、CaD、ACTG2表达的影响。结果:1.痰气交阻证、瘀血内结证、津亏热结证和气虚阳微证食管癌病人的发病年龄不同,痰气交阻证病人发病年龄为60.6±8.1岁,瘀血内结证病人发病年龄为61.5±11.6岁,津亏热结证病人发病年龄为65.6±8.1岁,气虚阳微证病人发病年龄为68.4±7.3岁。2.痰气交阻证、瘀血内结证、津亏热结证和气虚阳微证组织中分别有自身抗原CK13、CaD、ACTG2和CK16的表达,抗原自身抗体可以用于检测食管癌组织自身抗原表达;3.CaD在食管癌病人癌组织中表达阳性率和阳性表达水平高于正常组织和癌旁组织,其癌旁组织中表达阳性率和阳性表达水平高于正常组织,CaD在食管癌病人正常组织、癌旁组织、癌组织中表达阳性率分别为50.9%、62.2%、72.8%,阳性表达水平分别为0.0085±0.0048、0.0107±0.0056、0.0177±0.0103;CK16、ACTG2在癌组织中的阳性表达水平高于正常组织和癌旁组织,癌旁组织中阳性表达水平高于正常组织,CK16在食管癌病人正常组织、癌旁组织、癌组织中阳性表达水平分别为0.0076±0.0033、0.0158±0.0065、0.0356±0.0165,ACTG2在食管癌病人正常组织、癌旁组织、癌组织中阳性表达水平分别为0.0091±0.0039、0.0136±0.0043、0.0214±0.0110;CK13在癌组织中的阳性表达水平低于正常组织和癌旁组织,癌旁组织中阳性表达表达水平低于正常组织,CK13在食管癌病人正常组织、癌旁组织、癌组织中阳性表达水平分别为0.2053±0.0311、0.1633±0.0280、0.0412±0.0239;4.食管癌病人癌组织CK13、CK16、CaD、ACTG2的表达在四种证候之间存在差异:瘀血内结证CaD阳性率为100%(6/6),其阳性表达水平为0.0364±0.0051,高于痰气交阻证(29/34,0.0168±0.0088)、津亏热结证(5/8,0.0159±0.0045)和气虚阳微证(5/7,0.0166±0.0089);津亏热结证ACTG2阳性表达水平为0.0346±0.0100,高于痰气交阻证0.0201±0.0106、瘀血内结证0.0244±0.0043和气虚阳微证0.0235±0.0077;气虚阳微证CK16阳性表达水平为0.0595±0.0131,明显高于痰气交阻证0.0325±0.0167、瘀血内结证0.0227±0.0154、津亏热结证0.0393±0.0068;气虚阳微证CK13阳性表达水平为0.0174±0.0058,低于痰气交阻证0.0452±0.0292、瘀血内结证0.0385±0.0209和津亏热结证0.0330±0.0251;CK13、CK16、CaD、ACTG2在食管癌病人正常组织和癌旁组织中表达的证候之间无差异;5.清热化痰方及其清热和化痰两个拆方对食管癌细胞EC109、EC9706和TE-1的增殖均存在抑制作用,抑制作用从强到弱依次为全方组、清热组、化痰组;6.清热化痰方及其清热和化痰两个拆方对食管癌细胞EC109、EC9706和TE-1细胞周期均有影响:与空白对照组比较,EC109、TE-1细胞的清热组、化痰组和全方组S期细胞均减少,其中化痰组变化最明显;EC9706细胞清热组S期细胞增加明显,G2期细胞减少明显,化痰组G1期细胞减少明显,G2期细胞增加明显,全方组G1期细胞增加明显,G2期细胞减少明显。7.清热化痰方及其清热和化痰两个拆方可以不同程度地调节食管癌细胞EC109、EC9706和TE-1中CK13、CK16、CaD、ACTG2表达,用药组CK13表达高于空白组,用药组CK16、CaD、ACTG2表达低于空白组。结论:1.食管癌病人癌组织均表达有自身抗原CK13、CK16、CaD、ACTG2,其中CK16、CaD、ACTG2在食管癌病变过程中表达上升,CK13表达下降;2.在食管癌不同证候病变过程中CK13、CK16、CaD、ACTG2均有改变,其中痰气交阻证CK13表达改变突出,瘀血内结证CaD表达改变突出,津亏热结证ACTG2表达改变突出,气虚阳微证CK16表达改变突出;3.清热化痰方及其清热和化痰两个拆方对食管癌细胞Eca109、EC9706、TE-1增殖的抑制作用存在剂量依赖和时间依赖关系;4.清热化痰方及其清热和化痰两个拆方对食管癌细胞Eca109、EC9706、TE-1的细胞周期有影响,其中Eca109、TE-1细胞各药物组主要作用于S期,EC9706主要作用于S期和G2期;5.清热化痰方及其清热和化痰两个拆方抑制食管癌细胞Eca109、EC9706和TE-1增殖与调节CK13、CK16、CaD、ACTG2蛋白表达相关为清热化痰方治疗食管癌提供了分子依据;6.食管癌痰阻、瘀结、津亏、气虚病机与自身抗原CK13、CK16、CaD、ACTG2表达改变相关。
[Abstract]:Objective: to analyze the expression and distribution of CK13, CK16, CaD and ACTG2 in the four syndromes of esophageal cancer, and to explore the regulation of the two dismantling prescriptions of clearing heat and phlegm and its heat clearing and resolving phlegm on the proliferation of esophageal cancer cells and the expression of their own antigen, for the molecular pathogenesis and clinical medication of the esophageal cancer syndrome. Methods: 1. Western blot analysis of esophageal cancer tissues was compared with the autoantigen antibody of esophageal cancer and the patient's own serum; 2. the expression of CK13, CK16, CaD, ACTG2 in four kinds of normal tissues of esophageal carcinoma, the expression of the para cancerous tissue and cancer tissues were analyzed by immunohistochemistry; 3. the MTT method was used to detect the heat clearing and eliminating phlegm, and the clearing heat and phlegm of the clearing heat and phlegm were detected by MTT method. The effects of two dismantling sides on the proliferation of EC109, EC9706 and TE-1 cells in esophageal cancer cells; 4. the effects of two dismantling sides of clearing heat and eliminating phlegm and clearing heat and phlegm on the cycle of EC109, EC9706 and TE-1 cells in esophageal cancer cells were analyzed by flow cytometry; 5. using Western blot to analyze the two dismantling sides of clearing heat and eliminating phlegm and eliminating phlegm and clearing heat and eliminating phlegm. The effect of EC109, EC9706 and TE-1 CK13, CK16, CaD, ACTG2 expression. Results: 1. sputum obstruction syndrome, blood stasis syndrome, Qi deficiency syndrome and Qi deficiency syndrome of esophageal cancer patients are different, the age of the patients with sputum resistance syndrome is 60.6 + 8.1 years old, the age of the patients with blood stasis syndrome is 61.5 + 11.6 years, and the patients with Tianjin deficiency heat syndrome Age is 65.6 + 8.1 years old, the age of qi deficiency syndrome patients is 68.4 + 7.3 years old.2. sputum resistance syndrome, blood stasis syndrome, Zimi heat syndrome and Qi deficiency syndrome, there are self antigens CK13, CaD, ACTG2 and CK16 expression, the antigen autoantibody can be used to detect the expression of self antigen of esophageal cancer tissue; 3.CaD in the cancer group of esophageal cancer patients The positive rate and positive expression level in the tissue were higher than that of the normal tissue and para cancerous tissue. The positive rate and positive expression level in the paracancerous tissues were higher than those of the normal tissue. The positive rates of CaD in normal tissues, para cancer tissues and cancer tissues of the patients with esophageal cancer were 50.9%, 62.2%, 72.8%, respectively, and the positive expression level was 0.0085 + 0.0048,0.0107, respectively. The positive expression level of ACTG2 in the cancer tissues was higher than that of normal tissue and para cancerous tissue. The positive expression level of the paracancerous tissues was higher than that of the normal tissue. The positive expression level of CK16 was 0.0076 + 0.0033,0.0158 + 0.0065,0.0356 + 0.0165 in the normal tissue, para cancer tissue and cancer tissue of the patients with esophageal cancer, and ACTG2 was in the diet, respectively. The positive expression level of ACTG2 in the cancer tissue was higher than that in the normal tissue. The positive expression level was 0.0091 + 0.0039,0.0136 + 0.0043,0.0214 + 0.0110 in normal tissue, para cancer tissue and cancer tissue in cancer patients. The positive expression level of CK13 in cancer tissues was lower than that of normal tissue and para cancer tissue. The positive expression level in para cancerous tissues was lower than that of normal tissue. CK13 was in normal tissue of the patients with esophageal cancer and in the para cancer group. The positive expression level in the carcinoma tissue was 0.2053 + 0.0311,0.1633 + 0.0280,0.0412 + 0.0239 respectively. The expression of CK13, CK16, CaD and ACTG2 in the carcinoma tissues of 4. patients with esophageal cancer was different between the four syndromes: the positive rate of CaD in the blood stasis syndrome was 100% (6/6), and the positive expression level was 0.0364 + 0.0051, which was higher than that of the sputum resistance syndrome (29/34,0.0168 + 0.0088). The syndrome of Jin deficiency heat syndrome (5/8,0.0159 + 0.0045) and Qi deficiency Yang Micro syndrome (5/7,0.0166 + 0.0089); the positive expression level of ACTG2 in Tianjin deficiency heat syndrome was 0.0346 + 0.0100, higher than that of sputum obstruction syndrome (0.0201 + 0.0106, 0.0244 + 0.0043 and Qi deficiency Yang Micro syndrome 0.0235 + 0.0077), and the positive expression level of qi deficiency Yang microsyndrome was 0.0595 + 0.0131, obviously higher than phlegm. The syndrome of gas crossing was 0.0325 + 0.0167, the syndrome in blood stasis was 0.0227 + 0.0154, the syndrome of Jin deficiency fever was 0.0393 + 0.0068, the positive expression level of CK13 in Qi deficiency syndrome was 0.0174 + 0.0058, lower than that of the sputum cross resistance syndrome, 0.0452 + 0.0292, the syndrome in stasis syndrome 0.0385 + 0.0209 and Jin deficiency syndrome 0.0330 + 0.0251, and CK13, CK16, CaD, ACTG2 in the normal tissue and para cancer of the patients with esophageal cancer. There were no differences between the syndromes expressed in the tissues; 5. the two dismantling sides of clearing heat and eliminating phlegm and clearing heat and resolving phlegm had inhibitory effect on the proliferation of EC109, EC9706 and TE-1 of esophageal cancer cells. The inhibitory effect was from strong to weak to the whole group, the clearing heat group and the phlegm group; 6. clearing heat and Phlegm Recipe and its clearing heat and phlegm two dismantling prescription on the esophageal cancer cells EC109, EC97 The cell cycle of 06 and TE-1 had an effect: compared with the blank control group, the cells in the EC109 and TE-1 cells were reduced in the clearing heat group, the phlegm group and the whole group were reduced in the S stage, of which the phlegm group had the most obvious change, the S phase cells in the EC9706 cell heat clearing group increased obviously, the G2 phase cells decreased obviously, the cell decrease in the phlegm group G1 phase decreased obviously, the G2 phase cells increased obviously, the G1 period of the whole group G1 period was more obvious. The cell increase obviously, G2 stage cell decrease obviously.7. clearing heat and phlegm prescription and two dismantling sides of clearing heat and phlegm can adjust esophageal cancer cells EC109, EC9706 and TE-1 CK13, CK16, CaD, ACTG2 expression, the expression of CK13 in the drug group is higher than that of the blank group, the drug group CK16, CaD, is lower than the blank group. Conclusion: all of the cancer tissues of the patients with esophageal cancer are all cancer tissue The expression of its own antigen CK13, CK16, CaD, ACTG2, CK16, CaD, ACTG2 in the process of esophageal cancer, the expression of CK13 decreased; 2. in the process of esophageal cancer, CK13, CK16, CaD, ACTG2 all changed. The change of CK16 expression was prominent. 3. the two dismantling prescriptions of clearing heat and eliminating phlegm and clearing heat and resolving phlegm had dose dependence and time dependence on the proliferation of Eca109, EC9706, and TE-1 in esophageal cancer cells; 4. the cell cycle of Eca109, EC9706 and TE-1 in the carcinoma cells of the esophagus and its clearing heat and the phlegm and its clearing heat and the resolving phlegm. Among them, Eca109, TE-1 cells were mainly used in S phase, EC9706 mainly acted on S and G2 phase; 5. the two dismantling sides of clearing heat and eliminating phlegm and clearing heat and resolving phlegm to inhibit the Eca109, EC9706 and TE-1 proliferation and regulation CK13, CK16, CaD, and CaD, and the expression phase for the treatment of esophageal cancer with molecular basis; 6. The pathogenesis of esophageal cancer phlegm blockage, stasis, deficiency of Qi and deficiency of Qi is related to the expression of CK13, CK16, CaD and ACTG2.
【学位授予单位】：河南中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R273

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