消痰散结方联合放疗对肺癌大...免疫功能及肿瘤标志物的影响_刘香来.pdf

中国中医急症 2023年1月第32卷第1期JETCM.Jan.2023，Vol.32，No.1消痰散结方联合放疗对肺癌大鼠癌细胞凋亡、免疫功能及肿瘤标志物的影响刘香来1刘继微2李海川1李明利3（1.河北省廊坊市中医医院，河北 廊坊 065000 2.河北省廊坊爱德堡医院，河北 廊坊 065000；3.河北省邯郸市第一医院，河北 邯郸 056000）中图分类号：R285.5文献标志码：A文章编号：1004-745X（2023）01-0068-05doi：10.3969/j.issn.1004-745X.2023.01.015【摘要】目的 观察消痰散结方联合放疗对肺癌大鼠癌细胞凋亡、免疫功能及肿瘤标志物影响。方法 选取SD雄性大鼠进行肺癌建模，建模成功后随机分为模型组、消痰散结方组、放疗组、消痰散结方联合放疗组，另设正常组，每组10只。中药组予消痰散结方煎剂0.4 mL/只灌胃，放疗组给予放射治疗，联合组给予消痰散结方联合放疗治疗，观察并记录大鼠体质量、肿瘤质量及肿瘤体积，HE染色法检测组织病理形态，TUNEL法检测癌细胞凋亡，流式细胞仪检测免疫功能相关指标，ELISA法检测肿瘤标志物。结果 与正常组相比，模型组体质量、血清CD3+、CD4+细胞含量显著降低（P0.05），肿瘤质量、肿瘤体积、CD8+、NKT细胞、CEA、NSE、CA125含量显著升高（P0.05）；与模型组相比，中药组体质量、癌细胞凋亡、血清CD3+、CD4+细胞含量明显升高（P0.05），肿瘤质量、肿瘤体积、CD8+、NKT细胞、CEA、NSE、CA125含量显著降低（P0.05）；与放疗组和中药组相比，联合组体质量、癌细胞凋亡、血清CD3+、CD4+细胞含量明显升高（P 0.05），肿瘤质量、肿瘤体积、CD8+、NKT细胞、CEA、NSE、CA125含量显著降低（P 0.05）。正常组肺组织结构、肺泡正常完整，排列整齐，间质均匀且无充血；模型组可见肺泡囊明显扩张，肺间质明显增厚，纤毛上皮细胞出现明显破损，可见大量炎性细胞及癌细胞浸润，核仁明显，排列紊乱并伴有腺样或乳头状结构；与模型组比较，中药组、放疗组、联合组病理结构明显改善，炎性细胞及癌细胞明显减少。结论 消痰散结方联合放疗可显著促进肺癌大鼠癌细胞凋亡，提高其免疫功能，并降低肿瘤标志物的水平。【关键词】肺癌放疗消痰散结方癌细胞凋亡免疫功能肿瘤标志物大鼠Effect of Xiaotan Sanjie Decoction Combined with Radiotherapy on Apoptosis，Immune Function andTumor Markers of Lung Cancer Cells in RatsLiu Xianglai，Liu Jiwei，Li Haichuan，Li Mingli.LangfangHospital of Traditional Chinese Medicine，Hebei，Langfang 065000，China.【Abstract】Objective：To investigate the effects of Xiaotan Sanjie Decoction combined with radiotherapy onapoptosis，immune function and tumor markers of lung cancer cells in rats.Methods：Lung cancer models was established in SD male rats.After successful modeling，they were randomly divided into model group，Xiaotan SanjieDecoction group，radiotherapy group，Xiaotan Sanjie Decoction combined with radiotherapy group，and normalgroup，with 10 rats in each group.Xiaotan Sanjie Decoction group was given 0.4 mL of Xiaotan Sanjie Decoction，radiotherapy group was given radiotherapy，and combination group was given Xiaotan Sanjie Decoction combinedwith radiotherapy.The body mass，tumor mass and tumor volume of rats were observed and recorded，Histopathological morphology was detected by HE staining，apoptosis of cancer cells was detected by TUNEL，immune function related indicators were detected by flow cytometry，and tumor markers were detected by ELISA.Results：Compared with normal group，the body weight，serum contents of CD3+and CD4+cells in model group were significantly decreased（P 0.05），tumor weight，tumor volume，contents of CD8+，NKT cells，CEA，NSE and CA125were significantly increased（P 0.05）.Compared with model group，the body weight，apoptosis of cancer cells，serum contents of CD3+and CD4+cells in Xiaotan Sanjie Decoction group were significantly increased（P0.05），tumor weight，tumor volume，contents of CD8+，NKT cells，CEA，NSE and CA125 were significantly decreased（P 0.05）.Compared with radiotherapy group，the body weight，apoptosis ofcancer cells，serum contents of CD3+and CD4+cells in combination group were significantly increased（P0.05），while tumor weight，tumor volume，serum contents of CD8+，NKT cells，CEA，NSE and CA125 were significantlydecreased（P 0.05）.In normal group，the structure of the lung tissue and alveoli were normal and intact，arranged in an orderly fashion，the interstitium was even and without congestion；in model group，the alveolar sacwas obviously dilated，the lung interstitium was obviously thickened，the ciliated epithelial cells appeared obviously broken，a large number of inflammatory cells and cancer cell infiltration were visible，the nucleolus was obvious，arranged in a disorder and accompanied by adenoid or papillary structure.Compared with model group，the pathological structure of Xiaotan Sanjie Decoction groups，radiotherapy groups，and Xiaotan Sanjie Decoction combinedwith radiotherapy group was significantly improved，and the inflammatory cells and cancer cells were obviously reduced.Conclusion：Xiaotan Sanjie Decoction combined with radiotherapy can significantly promote the apoptosisof lung cancer cells in rats，improve the immune function，and reduce the level of tumor markers.【Key words】Lung cancer；Radiotherapy；Xiaotan Sanjie Decoction；Apoptosis of cancer cells；Immune function；Tumor markers；Rats-69中国中医急症 2023年1月第32卷第1期JETCM.Jan.2023，Vol.32，No.1计算，中药组每只大鼠灌胃4 mL消痰散结方煎剂，每天1次，共28 d。同时，放疗组采用高能直线加速器，在200 cGy/min的固定剂量下进行6 MV-X射线放射治疗，2 d 1次，共28 d。联合组给予消痰散结方联合放疗治疗，方法与中药组和放疗组相同，给药后24 h进行放射治疗。正常组、模型组每日给予同体积生理盐水灌胃。1.5标本采集与检测给药结束24 h后，称重后腹腔注射3%戊巴比妥钠麻醉处死大鼠，采集肺部动脉血，然后取双肺及肿瘤组织。肺血离心后取上清液于冰箱中密封保存。双肺用4%的多聚甲醛固定96 h，生理盐水漂洗干净，放入冰箱中密封保存。肿瘤组织称重，称重后对其长径及短径进行测量，按照公式 （长径短径2）2 计算瘤体体积。1.5.1肺病理组织HE染色取各组右肺组织，常规石蜡包埋、切片、脱蜡、复水后进行HE染色，染色完成后除去多余染液，进行脱水、二甲苯透明和中性树胶封片处理，在光学显微镜下随机选取3个视野进行组织病理学观察。1.5.2末端脱氧核苷酸标记法（TUNEL）检测癌细胞凋亡取各组左肺组织进行常规石蜡包埋、切片、脱蜡后，将切片置于3%过氧化氢中浸泡10 min，PBS洗涤3次，加入蛋白酶K后进行室温孵育10 min，PBS洗涤3次，然后缓冲液标记2 h，封闭液封闭30 min，甩掉封闭液后，加入稀释的生物素化抗地高辛抗体（1 100），在37 温育箱中反应30 min，加入稀释的SABC-FITC抗体（1 100），在37 温育箱中反应30 min，PBS洗涤4次后，用DAPI染色液进行染色，染色完成后用抗荧光衰减封片剂封片。在荧光显微镜下观察，黄绿色荧光颗粒在细胞核中的为凋亡细胞，随机选择5个视野下的细胞计算凋亡率，凋亡率=凋亡细胞数总细胞数100%。1.5.3流式细胞仪检测血清免疫功能相关指标取大鼠血清，加入10 L CD3+、10 L CD4+、10 L CD8+、10 L NKT抗体和50 L EDTA抗凝血，震荡混合均匀后温室避光孵育20 min，