半夏瀉心湯聯合IL-12轉染骨髓間充質干細胞對胃癌荷瘤裸鼠抑瘤作用研究

崔國寧　劉喜平　陳嘉慧　崔樹婷　譚霞

摘要：目的? 觀察半夏瀉心湯聯合IL-12轉染骨髓間充質干細胞（BMSCs）對胃癌荷瘤裸鼠的抑瘤作用。方法? 取培養第3代的BMSCs，采用LV5空載慢病毒及LV5 IL-12過表達慢病毒轉染BMSCs細胞，分別于轉染48、72 h后，倒置熒光顯微鏡觀察，經嘌呤霉素篩選穩定轉染BMSCs，RT-qPCR檢測BMSCs中IL-12的表達。Balb/C裸鼠腋窩皮下接種人胃癌BCG-823細胞建立胃癌荷瘤裸鼠模型，實驗分為成瘤組、空載組、轉染組、藥物組、聯合組。空載組、轉染組和聯合組經尾靜脈注射0.2 mL含1×10⁷/mL轉染慢病毒的BMSCs懸液，藥物組和聯合組給予半夏瀉心湯27.43 g/kg灌胃，每次0.5 mL，成瘤組給予等量生理鹽水灌胃，隔日測量并計算瘤體體積，連續干預13 d，取瘤體組織，計算抑瘤率。結果? 倒置熒光顯微鏡下穩定轉染病毒的BMSCs呈不規則形、星形，蜂窩狀綠色熒光分布，經RT-qPCR驗證，轉染BMSCs的IL-12 mRNA表達升高，與正常BMSCs、空載BMSCs比較差異有統計學意義（P<0.05）;與成瘤組、空載組比較，轉染組、藥物組及聯合組瘤體積及瘤質量增加較小（P<0.05）;與轉染組、藥物組比較，聯合組瘤體積及瘤質量增加最小（P<0.05）;與空載組比較，轉染組、藥物組及聯合組抑瘤率明顯升高（P<0.05）;與轉染組、藥物組比較，聯合組抑瘤率明顯升高（P<0.05）。結論? 半夏瀉心湯及IL-12轉染的BMSCs對胃癌荷瘤裸鼠均有一定的抑瘤作用，二者聯合具有協同增效作用。

關鍵詞：半夏瀉心湯;骨髓間充質干細胞;白細胞介素-12;裸鼠

中圖分類號：R285.5 ???文獻標識碼：A??? 文章編號：1005-5304（2020）01-0039-06

DOI：10.3969/j.issn.1005-5304.201906381

Study on Antitumor Effects of Banxia Xiexin Decoction Combined with IL-12 Transfected BMSCs on Nude Mice with Gastric Cancer

CUI Guoning， LIU Xiping， CHEN Jiahui， CUI Shuting， TAN Xia

Gansu University of Chinese Medicine， Lanzhou 730000， China

Abstract： Objective To observe the antitumor effects of Banxia Xiexin Decoction combined with IL-12 transfected BMSCs on nude mice with gastric cancer. Methods BMSCs of the third generation were cultured and transfected by LV5 no-load lentiviruses and IL-12 overexpression lentiviruses into BMSCs. The expression of IL-12 in BMSCs was detected by RT-qPCR after transfected 48 hours and 72 hours through inverted fluorescence microscopy. A nude mouse model of gastric cancer was established by subcutaneous inoculation of human gastric cancer BCG-823 cells into the axilla of Balb/C nude mice. The model was divided into tumorigenic group， no-load group， transfection BMSCs， medicine group and combination group. No-load BMSCs， transfection group and combination group were injected 0.2 mL BMSCs suspension containing 1×10⁷/mL lentivirus via tail vein respectively. The medicine group and combination group were given Banxia Xiexin Decoction for gavage （27.43 g/kg， 0.5 mL each time）. The tumorigenic group was given the same dose of saline for gavage. Tumor volume of each group was measured and calculated every other day. After 13 days of continuous intervention， tumor tissue was taken to calculate the antitumor rate. Results BMSCs stably transfected with the virus showed irregular， star-shaped and honeycomb-like green fluorescence distribution under inverted fluorescence microscopy. The expression of IL-12

mRNA in the transfection BMSCs was increased by RT-qPCR， and there was statistical significance between the normal BMSCs and no-load BMSCs （P<0.05）. Compared with tumorigenic group and no-load group， the tumor mass and tumor weight of the transfection group， the medicine group and the combination group increased little （P<0.05）; Compared with the transfection group and the medicine group， the tumor mass and tumor weight of the combination group were the least （P<0.05）; Compared with the no-load group， the antitumor rates of the transfection group， the medicine group and the combination group significantly increased （P<0.05）. Compared with the transfection group and the medicine group， the antitumor rate of the combination group significantly increased （P<0.05）. Conclusion Banxia Xiexin Decoction combined with BMSCs transfected with IL-12 have certain anti-tumor effects on nude mice with gastric cancer， and their combination has a significant synergistic effect.

Keywords： Banxia Xiexin Decoction; BMSCs; IL-12; nude mice

骨髓間充質干細胞（bone marrow mesenchymal stem cells，BMSCs）是起源于中胚層的一種多能干細胞，具有腫瘤趨向特性，是腫瘤靶向治療首選種子細胞^[1]。IL-12是一種免疫刺激性細胞因子，被認為是細胞免疫應答反應中的初始因子，能激活T淋巴細胞、NK細胞，破壞腫瘤細胞的免疫逃逸，具有很強的抗腫瘤作用^[2]。轉染是真核細胞主動或被動導入外源的DNA片段，從而獲得新表型的過程。已有研究表明，通過轉染將IL-12導入BMSCs，使其過表達IL-12，對卵巢癌、結腸癌有顯著抑瘤作用^[3-4]。半夏瀉心湯是臨床防治胃癌的常用方劑，臨床療效確切^[5]。……