【作者】 易华；

【导师】 李杰芬；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2009， 博士

【摘要】 原发性肝癌是世界范围内发病率最高的恶性肿瘤之一,素有“癌王”之称。中国肝癌发病人数居世界首位,每年约23万人死于肝癌,其恶性程度高,预后差。使用基因疗法对付肿瘤已经成为中外科学家一个重要的选择。1989年—2003年6月,美国批准注册的基因治疗临床试验方案共523项,约占世界总数的80%,接受治疗病例数达到15000人,其中肿瘤基因治疗约占70%/。自杀基因疗法因存在旁观者效应的独特机制而成为具有广泛应用前景的肿瘤治疗新方法,但单纯应用不能完全治愈肿瘤,因而增强自杀基因治疗的效果成为研究热点。改善荷瘤机体的免疫状态是其增效措施之一,中药方药具有肯定的免疫药理作用,课题组前期以此为切入点开展研究,证实滋阴补肾经典复方六味地黄丸对肝癌HSV-tk/GCV自杀基因治疗具有增效作用,其机理与该方改善自杀基因抗肿瘤作用的炎症免疫微环境,增强自杀基因的旁观者效应有关。而GJIC介导的缝隙连接机制被认为是自杀基因旁观者效应的主要机制,鉴于中药方药具有多途径、多靶点的特点,该方对自杀基因治疗增效作用的机制也可能与缝隙连接机制相关。本课题拟对其进行研究,探讨六味地黄丸对自杀基因治疗的增效作用是否与其改善肝癌细胞GJIC功能相关,并进一步明确其机制。一、研究目的:本研究在前期工作的基础上,应用中药复方血清药理学的方法,以大鼠肝癌细胞CBRH7919为研究对象,初步探讨六味地黄丸对肝癌HSV-tk/GCV自杀基因治疗增效作用的缝隙连接机制,以期为临床自杀基因疗法联合中药复方的中西医结合治疗方案的建立提供实验数据、科学依据及工作基础。二、研究方法:1.免疫组化SABC法检测肝癌荷瘤小鼠肿瘤组织Cx26、Cx43、Cx32蛋白的表达:选用课题组前期实验保存的3批肝癌荷瘤小鼠肿瘤组织的石蜡标本（分为肿瘤模型组、自杀基因治疗组、六味地黄丸治疗组、自杀基因治疗联合六味地黄丸治疗组共4个组）,每组选取18个左右动物蜡块,常规切片DAB免疫组化染色,显微镜下随机选取5个高倍视野计数,根据显色强度和阳性细胞数两项指标判断阳性等级。2.四甲基偶氮唑盐（MTT法）观测六味地黄丸含药血清对自杀基因HSV-tk/GCV系统杀伤大鼠肝癌CBRH7919细胞的增效作用:选用前期构建的HSV-tk/GCV CBRH7919自杀基因治疗系统,确定丙氧鸟苷（GCV）工作浓度为39.2μmol/L、鼠血清添加量为10%（体积分数）;制备SD大鼠4d和8d六味地黄丸(32g·kg^-1·d^-1)灌胃含药血清和生理盐水灌胃的对照血清;大鼠肝癌细胞CBRH7919/tk^-及CBRH7919/10%tk⁺（tk⁺与tk^-细胞以1:9比例混合）细胞按3×10³个/孔密度接种96孔板,设对照血清组、GCV加对照血清组、1 0%tk⁺/GCV联合对照血清组、含药血清组、GCV加含药血清组及10%tk⁺/GCV联合含药血清组;8d含药血清组又再分为低剂量组、中剂量组和高剂量组,每组6个复孔,用完全培养基培养24h后,相应组分别加入空白血清或含药血清,孵育12h,加入GCV培养60h,MTT法检测细胞存活率,用Q值（实测药效与理论药效的比值）分析自杀基因系统与含药血清联合的相互作用是否具有协同性（0.85≤Q≤1.15为相加作用,Q≥1.15为协同作用）。3.检测六味地黄丸含药血清对CBRH7919细胞GJIC功能的影响:设血清对照组（10%鼠空白血清）、含药血清低剂量组（2.5%含药血清+7.5%对照血清）、中剂量组（5%含药血清+5%对照血清）和高剂量组（10%含药血清）。添加不同浓度的六味地黄丸含药血清孵育CBRH7919细胞4gh,采用预标记双染料传输技术及荧光光漂白恢复技术检测各组细胞染料传输功能即GJIC功能的状况。4.采用实时荧光定量RT-PCR技术检测六味地黄丸含药血清对CBRH7919细胞Cx26、Cx43、Cx32 mRNA表达的影响:设细胞对照组（不添加鼠血清）、血清对照组（10%鼠空白血清）、含药血清低剂量组（2.5%含药血清+7.5%空白血清）、中剂量组（5%含药血清+5%空白血清）和高剂量组（10%含药血清）。含药血清作用CBRH7919细胞48h,应用实时荧光定量RT-PCR染料法检测Cx26、Cx43、Cx32mRNA的相对表达量。以看家基因GAPDH作为实时荧光定量PCR反应的内参照,结果计算:Δct=Cx的ct均值—GAPDH的ct均值,然后取2^-ΔCt即代表某样品其初始cDNA的相对量。5.检测六味地黄丸含药血清对CBRH7919细胞Cx26、Cx43、Cx32蛋白表达的影响:六味地黄丸含药血清作用CBRH7919细胞48h,然后采用FITC间接免疫荧光染色,激光扫描共聚焦显微镜观察和流式细胞仪及Western-blot技术检测各组细胞Cx26、Cx43、Cx32蛋白的相对表达量。三、研究结果:1.免疫组化SABC法检测肝癌荷瘤小鼠肿瘤组织Cx26、Cx43、Cx32蛋白的表达,镜下观察阳性表达定位于细胞浆和细胞膜。Cx43在癌旁组织,特别是有纤维组织浸润的部位表达呈强阳性,而在癌结节中肿瘤细胞表达相对较弱;Cx26、Cx32在肿瘤细胞特别是脂肪组织浸润附近的癌组织中呈现强阳性表达。GCV治疗组三种Cx蛋白的表达与肿瘤模型组水平差异不大（P＞0.05）,而六味地黄丸治疗组和联合治疗组的蛋白表达均高于肿瘤模型组,其中联合组三种蛋白阳性表达与模型组的差异具有统计学意义（P＜0.05）。2.对照血清组、含药血清组、GCV加对照血清组、GCV加含药血清组细胞均未显示明显细胞毒性,表明在实验条件下血清和GCV对肿瘤细胞的生长无明显影响;自杀基因系统10%tk+/GCV加对照血清,及10%tk+/GCV联合不同浓度的含药血清组均具有明显的杀伤作用（P＜0.05,与空白血清组比较）,表明自杀基因系统具有生物学功能;10%tk+/GCV系统联合5%、10%含药血清组的细胞存活率均明显低于10%tk⁺/GCV加对照血清组及相对应的含药血清组,联合组的实际抑制率均显著高于理论抑制率,Q值分别为1.16、1.49,均大于1.15,说明其相互作用具有协同性,且随着含药血清浓度的增高Q值增大,协同作用增强。对照血清和含药血清浓度在体积分数20%以下时未显示明显细胞毒性;血清浓度为体积分数5%和7.5%时,自杀基因系统联合含药血清组与单独含药血清组及自杀基因系统加对照血清组比较,联合组对肿瘤细胞的杀伤作用均强于其他非联合组,细胞存活率均有显著性差异（P＜0.01）,而且自杀基因系统联合含药血清的作用具有明显的协同性（Q＞1.15）。3.六味地黄丸含药血清对CBRH7919细胞GJIC功能的影响:（1）流式细胞仪测定结果显示,CBRH7919细胞经六味地黄丸含药血清作用一定时间后,与对照组相比,六味地黄丸含药血清组的双阳性细胞比例（红色和绿色荧光皆阳性的细胞数占总细胞数的比例）逐渐升高,到48h可达32%,达到饱和状态后随着时间的延长双阳性细胞比例逐渐下降;48h检测三个剂量组双阳性细胞的比例,对照组为18.76±2.22%,低剂量组27.93±3.55%,中剂量组35.23±4.15%,高剂量组39.93±5.71%,随着六味地黄丸含药血清浓度的增加,双阳性细胞率逐渐增加（低剂量组P＜0.05,中高剂量组P＜0.01）,并呈现出一定的剂量依赖效应。（2）从荧光漂白恢复图像上发现,在强激光漂白前,各组CBRH7919细胞都被激发出较强的蓝绿色荧光。选择的受试漂白细胞经瞬间漂白后细胞内荧光强度明显变弱,随着时间的推移荧光逐渐恢复,至4 min时对照血清组的平均荧光恢复率为5.93±0.72%,而含药血清组的平均荧光恢复率明显高于对照组,并呈现出一定的浓度依赖趋势。由低到高剂量组分别为15.71±3.81%,38.34±2.64%（P＜0.01）,46.22±4.33%（P＜0.01）。4.六味地黄丸含药血清作用于CBRH7919细胞48h后,随着六味地黄丸含药血清浓度的增加,CBRH7919细胞中Cx26、Cx32和Cx43基因mRNA相对表达量逐渐增多,并呈现出一定的剂量依赖效应,而Cx32和Cx43基因mRNA的表达以中剂量组最明显。设对照血清组Cx mRNA表达量为1,含药血清各剂量组Cx26mRNA相对表达量分别为6.63±0.78、19.29±3.62、29.04±5.35,与对照组相比差异有统计学意义（P＜0.01）;Cx32 mRNA相对表达量分别为23.02±5.10、261.55±29.11、73.68±16.23,Cx43 mRNA相对表达量分别为1.53±0.84、6.75±1.71、4.09±0.98,中、高剂量组Cx32和Cx43 mRNA与对照组相比差异有统计学意义（P＜0.01,P＜0.05）。5.激光共聚焦显微镜观察可见,各组阴性对照组未见阳性细胞表达,细胞对照组和血清对照组CBRH7919细胞表达Cx26、Cx32及Cx43较少,阳性细胞的荧光强度较弱。阳性细胞荧光表达主要集中在胞浆,部分在胞膜,特别是细胞连接部位;六味地黄丸含药血清各浓度组CBRH7919细胞表达Cx 26、Cx32及Cx43较多,阳性细胞的荧光强度较强。含药血清组Cx 32蛋白在CBRH7919细胞膜上呈强阳性表达,特别是细胞连接部位,荧光沿其细胞膜呈颗粒状或细线状分布,胞浆部分也有表达。Cx 26和Cx43阳性表达主要集中在胞浆、细胞连接处、胞膜及核膜周边,荧光呈颗粒状或细线状分布。流式细胞仪结果显示,与细胞血清组相比,鼠血清对照组三种Cx膜蛋白表达差异无统计学意义（P＞0.05）;与对照血清组相比,各浓度含药血清组Cx26、Cx32和Cx43膜蛋白的表达量均显著增加,差异有统计学意义（P＜0.05或P＜0.01）,尤其是Cx32含药血清组阳性细胞率可达70%以上。且呈现一定的剂量效应依赖趋势,与低剂量组相比,含药血清中、高剂量组Cx32阳性细胞率差异有统计学意义（P＜0.05）。而Cx43和Cx32的阳性细胞率有增高的趋势,但尚无统计学差异。Western-blot测定结果显示,含药血清各浓度组显著可提高Cx26、Cx32蛋白的表达,并呈现明显的量效关系。四、结论:1.免疫组化SABC法检测肝癌荷瘤小鼠肿瘤组织Cx26、Cx43、Cx32蛋白的表达结果显示,与肿瘤模型组相比,六味地黄丸联合自杀基因治疗组能增强Cx26、Cx43、Cx32蛋白表达,尤其是促进Cx26的表达,提示六味地黄丸增强自杀基因旁观者效应的机制与缝隙连接相关。2.HSV—tk/GCV自杀基因治疗系统联合六味地黄丸对杀伤肝癌细胞具有协同增效作用。3.六味地黄丸含药血清具有上调大鼠肝癌细胞CBRH7919 GJIC功能的作用,并呈一定的剂量依赖关系。4.六味地黄丸含药血清能显著增加CBRH7919细胞Cx26、Cx32和Cx43总蛋白的表达,并能增多其在细胞膜的定位分布,尤其促进Cx32在细胞膜的表达。并显示出一定的剂量依赖效应。5.六味地黄丸含药血清能显著增加CBRH7919细胞Cx26、Cx32和Cx43mRNA的表达,从转录水平增强Cx的表达。综上所述,六味地黄丸可能通过调控大鼠肝癌CBRH7919细胞Cx26、Cx32Cx43蛋白及mRNA表达、促进Cx的成熟与定位分布,在一定程度上恢复了Cx介导的GJIC功能,重新建立细胞间缝隙连接,进而提高HSV-tk/GCV系统旁观者效应来实现对自杀基因系统杀伤肝癌细胞的协同性增效作用。更多还原

【Abstract】 Primary hepatic carcinoma（PHC） is one of the highest incidences of malignant tumor in the worldwide,known as "cancer King".The number of the patient of PHC in China ranks first in the world.Every year about 23 million people die of the PHC because of its high degree of malignancy and poor prognosis.Using the gene therapy against tumors has become an important choice for scientists all over the world to choose.1989 -2003 years,the United States approved the registration of clinical trials of gene therapy for a total of 523,representing about 80%of the total of the world. The number of the patient who has been treated has achieved tol5,000,including 70%of the patient who was treated by the gene therapy.The Suicide gene therapy has a wide application prospects to become the new treatment of cancer because of the unique bystander effect.But the Suicide gene therapy alone can not completely cure the tumor,thereby enhancing the effectiveness of suicide gene therapy become a research hotspot.To improve the immune state of the tumor-bearing body is one of the measures of its efficiency,and the Chinese medicine has a definite role in the immune pharmacology.The study group makes it as a starting point,confirming the synergy of Liu Wei Di Huang bolus on hepatocarcinoma HSV-tk/GCV suicide gene therapy.The mechanism may be related with the fact that Liu Wei Di Huang bolus enhance the bystander effect of suicide gene by improving the inflammatory and immune micro-environment where the suicide gene faint against the tumor.The gap junction mechanism mediated by GJIC is considered the main mechanism of suicide gene’s bystander effect.In view of Chinese herbs with multi-channel,multi-target characteristics,the synergy of Liu We Di Huang bolus on hepatocarcinoma suicide gene therapy may be related to the mechanism of gap junction.The subject of this study is to examine whether the synergy of Liu We Di Huang bolus on hepatocarcinoma suicide gene therapy is related to that it can improve the function of hepatocarcinoma cell’s GJIC,and further clarify its mechanism.Objective:on the basis of preliminary work,we study the rat’s hepatocarcinoma CBRH7919 cell to explore the gap junction mechanism of the synergy of Liu We Di Huang bolus on hepatocarcinoma HSV-tk/GCV suicide gene therapy,with a view to provide experimental data,scientific evidence and the basis of the work for the establishment of clinical suicide gene therapy combined with traditional Chinese medicine.Methods:1.Using the SABC immtmohistochemical assay to detect the expression of Cx26, Cx43,Cx32 in hepatocarcinoma tumor-bearing mice:We choose the paraffin-embedded tumor tissue of hepatocarcinoma tumor-bearing mice.The tumor tissue is divided into tumor model group,suicide gene therapy group,Liu We Di Huang bolus in treatment group,suicide gene therapy combined with Liu We Di Huang bolus in treatment group,a total of 4 groups.We selecte about 18 pieces of animal wax from each group,conventional biopsy DAB immunohistochemical staining,and then select 5 high power fields randomly under the microscope to count. According to the color intensity and the number of positive cells in the two indicators, then we determined positive rating.2 To examine the killing effect on rat’s hepatoma cells CBRH7919 by using medicated serum of Liu Wei Di Huang bolus combined HSV-tk/GCV suicide gene therapy system.Selecte the pre-built HSV-tk/GCV CBRH7919 suicide gene therapy system,make sure that the GCV’s working concentration is 39.2μmol/L;the mouse serum’s adjunction is 10%（volume fraction）;Medicated or non-medicated serums were obtained from SD rats which were treated with Liu Wei Di Huang Bolus（the dose was 32 g/kg.d）.CBRH7919/tk+ cells and CBRH7919/tk - cells were mixed in proportion of 1:9 and were used as 10%tk+/GCV target cells.CBRH7919 cells were sorted into following experimental groups:non-medicated serum group（10% non-medicated serum,final concentration,v:v）,GCV plus non-medicated serum group,non-medicated serum plus 10%tk⁺/GCV group,low-dose medicated serum group（2.5%medicated serum+ 7.5%non-medicated serum,final concentration,v:v）, GCV plus low-dose medicated serum group,low-dose medicated serum+10% tk+/GCV group,mid-dose medicated serum group（5%medicated serum+ 5% non-medicated serum,final concentration,v:v）,GCV plus mid-dose medicated serum group,mid-dose medicated serum plus 10%tk⁺/GCV group,high-dose medicated serum group（10%medicated serum,final concentration,v:v）,GCV plus high-dose medicated serum group,high-dose medicated serum plus 10%tk⁺/GCV group. CBRH7919/tk^- cells or 10%tk⁺ cells were seeded in quadruplicate in 96-wells plates at a density of 3×103/well,grown in complete medium for 24 hours,,treated with medicated or non-medicated serum for 12 hours and sequentially with GCV at 39.2μmol/L for another 60 hours.The viability of cells was determined by MTT assay. Q value analysis,the ratio of the actual effect of combined treatment to its theoretical effect,was used to value the synergistic effect of the medicated serums on the suicide gene system.The effect was classified into three categories:antagonistic effect（Q≤0.85）,additive effect（0.85≤Q＜1.15）,and synergistic effect（Q≥1.15）.3.To examine the influence of the herbs serum of Liu Wei Di Huang Bolus on the GJIC function of CBRH7919 cell:CBRH7919 cells were sorted into following experimental groups:non-medicated serum group（10%non-medicated serum,final concentration,v:v）,low-dose medicated serum group（2.5%medicated serum+ 7.5% non-medicated serum,final concentration,v:v）,mid-dose medicated serum group（5% medicated serum+ 5%non-medicated serum,final concentration,v:v）,high-dose medicated serum group（10%medicated serum,final concentration,v:v）,treated with medicated serum of different concentration for 48h.The GJIC function is detected by the preloading and dye transfer and the fluorescence photo bleaching recover technique.4.To examine the influence of the herbs serum of Liu Wei Di Huang Bolus on the expression of Cx26,Cx43,Cx32 and mRNA on rats hepatoma cell line CBRH7919 by using Real-time fluorescent quantitative PCR assay:CBRH7919 cells were sorted into following experimental groups:non-cell group（without serum）,non-medicated serum group（10%non-medicated serum,final concentration,v:v）,low-dose medicated serum group（2.5%medicated serum+ 7.5%non-medicated serum,final concentration,v:v）,mid-dose medicated serum group（5%medicated serum+ 5% non-medicated serum,final concentration,v:v）,high-dose medicated serum group（10%medicated serum,final concentration,v:v）,treated with medicated serum of different concentration for 48h.The expression of Cx26,Cx43,Cx32 and mRNA is detected by Real-time fluorescent quantitative PCR assay.Make the house-keeping gene GAPDH as the quod vide of the fluorescent quantitation RT-PCR reaction,result calculationΔct=ct’s mean of Cx-ct’s mean of GAPDH,then make 2^-ΔCt to present the relative amount of primary cDNA.5 To examine the influence of the herbs serum of Liu Wei Di Huang Bolus on the the expression of Cx26,Cx43,Cx32 and mRNA on rats hepatoma cell line CBRH7919. The CBRH7919 cell is treated with medicated serum of different concentration for 48h.The expression of Cx26,Cx43,Cx32 and mRNA is detected by western blot, indirect immunofluorescence assay（FITC） and flow cytometry.Results:1.The expression of Cx26,Cx43,Cx32 in hepatocarcinoma tumor-bearing mice is detected by the SABC immunohistochemical assay,the positive expression were observed located in cytoplasm and cell membrane under the microscope.Cx43 in tumour adjacent tissues,especially the site which is infiltrated by fibrous tissue was strongly positive expression,but cancer cells in the tumor nodules are relatively weak expression.Cx26,Cx32 in tumor cell,especially near the tumour tissues which is infiltrated by adipose tissue show strong positive expression.The expression of Cx in GCV in treatment group is similar to that in tumor model group（P＞0.05）,but The expression of Cx in Liu We Di Huang bolus in treatment group and suicide gene therapy combined with Liu We Di Huang bolus in treatment group is obviously stronger than that in tumor model group.The differences of expression of Cx between suicide gene therapy combined with Liu We Di Huang bolus in treatment group and tumor model group shows statistical significance.2.The cell of non-medicated serum group,medicated serum group,GCV plus non-medicated serum group,GCV plus medicated serum group shows no obvious cytotoxicity.Under the experimental conditions show that the serum and GCV on the growth of tumor cells had no significant effect;GCV plus low-dose medicated serum group,GCV plus different medicated serum groups both has obvious killing effect（P <0.05,compared with non-medicated serum group）,Show that the suicide gene system has a biological function.The cell survival of mid-dose medicated serum plus 10%tk+/GCV group and high-dose medicated serum plus 10%tk+/GCV group is obvious lower than that of non-medicated serum plus 10%tk+/GCV group and corresponding medicated serum group;The actual inhibition rate of joint Group is significantly higher than the theoretical inhibition rate.Q values is 1.16、1.49,both larger than 1.15,shows that its interaction has the synergy,with the concentration of medicated serum increasing,Q values increases and synergies enhances.Non-medicated serum group and medicated serum group below 20%volume fraction do not show obvious cytotoxicity.When the volume fraction of the serum concentration is 5%and 7.5%,compared with seperate medicated serum group and GCV plus non-medicated serum group,GCV plus medicated serum group has a stronger killing effect on tumor cells,the cell survival rate were significantly different （P<0.01）,and the effect of GCV combining with medicated serum has obvious synergies（Q>1.15）.3.The influence of the herbs serum of Liu Wei Di Huang Bolus on the GJIC function of CBRH7919 cell:（1）The result of flow cytometry shows that,with treated with medicated serum for some time,compared with the control group,the proportion of the double positive cells gradually increases.It can go up to 32%after 48h.After saturated,with the time,the proportion of double-positive cells gradually decrease.After 48h,detect the proportion of double positive cells Of the three-dose group,the result is:the control group is 18.76±2.22%,low-dose medicated serum group is 27.93±3.55%,mid-dose medicated serum group is 35.23±4.15%,high-dose medicated serum group is 39.93±5.71%.With medicated serum concentration increased,the rate of double-positive cells gradually increase（low-dose group P<0.05;mid-dose group, high-dose group P<0.01）,and shows a certain degree of dose-dependent effect.（2） Found from the image of fluorescence recovery after photobleaching （FRAP）,before strong photobleaching,CBRH7919 cells in each group were inspired to produce a strong blue-green fluorescence.The fluorescence intensity of chooesn cell is significantly weaker after transient bleaching.Over time,the fluorescence gradually returns.After 4 min,the average fluorescence recovery rate of the non-medicated serum group is 5.93±0.72.But the mean fluorescence recovery rate of medicated serum group was significantly higher than that of non-medicated serum group,and showing a certain degree of concentration-dependent trend.From low to high dose group,fluorescence intensity is 15.71±3.81%,38.34±2.64%（P＜0.01）,46.22±4.33%（P＜0.01）.4.After being treated with medicated serum for 48h,with medicated serum concentration increased,the relative expression of Cx26,Cx32 and Cx43,mRNA in CBRH7919 cells gradually increase and show a dose-dependent effect.But the expression of Cx32 and Cx43,mRNA is most obvious in mid-dose medicated serum group.Make the expression of Cx mRNA as 1,from low to high dose group,the relative expression of Cx26mRNA is 6.63±0.78,19.29±3.62,29.04±5.35.Compared with the control group,differences is statistically significant（P＜0.01）.The relative expression ofCx32 Mrna is 23.02±5.10,261.55±29.11,73.68±16.23,and the relative expression of Cx43 is 1.53±0.84,6.75±1.71,4.09±0.98.The different of the expression of Cx32 and Cx43 mRNA between mid,high-dose medicated serum group and the control group is statistically significant（P＜0.01,P＜0.05）.5.Found from the result of laser confocal microscopy,each negative control group show no positive cells,CBRH7919 cells of no-cell group and non-medicated serum group expressed Cx26,Cx32 and Cx43 less,the fluorescence intensity of positive cells is less.The expression of fluorescent-positive cells is mainly located in the cytoplasm, some in the membrane,in particular parts of cell junction.CBRH7919 cells of medicated serum group expressed Cx26,Cx32 and Cx43 more,the fluorescence intensity of positive cells is stronger.The expressure of Cx 32 in CBRH7919 cell membrane is strongly positive,especially at the cell junction site.Fluorescence was granular or thread-like distributed along its cell membrane,partly cytoplasmic.The expression of Cx26 and Cx43 was mainly concentrated in the cytoplasm,cell junction, membrane and nuclear membrane surrounding,and the fluorescence was granular or thread-like distribution.Found from the result of flow cytometry,compared with no-cell group,the different of the expression of Cx in non-medicated serum group is no significant （P＞0.05）;Compared with non-medicated serum group,The expression of Cx26, Cx32和Cx43 increase significantly,the difference was statistically significant（P＜0.05或P＜0.01）,especially the proportion of the positive cells in Cx32 medicated serum group is up to 70%,and has a certain dose-response trend.Compared with low-medicated serum group,the difference of the proportion of Cx32-positive cells between mid-medicated serum group and high-medicated serum group is statistically significant（P＜0.05）.The proportion of Cx43 and Cx32 positive cells is in the trend of increasing,but there is no statistical difference.Found from the result of Western-blot,medicated serum can significantly improve the expression ofCx26,Cx32,and shows a clear dose-effect relationship.Conclusion:1.Using the SABC immunohistochemical assay to detect the expression of Cx26, Cx43,Cx32 in hepatocarcinoma tumor-bearing mice.From the result,compared with tumor model group,suicide gene therapy combined with Liu We Di Huang bolus in treatment group can significantly improve the expression of Cx26,Cx43,Cx32, expecially Cx26.It is Prompte that the synergy of Liu Wei Di Huang bolus on Suicide gene bystander effect is related to gap junction.2.Liu Wei Di Huang bolus combined HSV-tk/GCV suicide gene therapy system has Synergies on killing tumour cells.3.Liu Wei Di Huang bolus medicated serum can improve the function of hepatocarcinoma cell’s GJIC,and shows a certain degree of dose-response effect.4.Liu Wei Di Huang bolus medicated serum can increase the expression of Cx26, Cx43,Cx32 in CBRH7919 cell,and increase their localization in the cell membrane, expecially Cx32.It shows a certain degree of dose-response effect.5.Liu Wei Di Huang bolus medicated serum can increase the expression of Cx26, Cx32 and Cx43 mRNA.It increases the expression of Cx from the transcriptional level.In conclusion,Liu Wei Di Huang bolus may promote the expression,maturity, distribution and location of the Cx in CBRH7919 cell.Through this way,it can recover the the Cx-mediated GJIC function to a certain extent,re-establish the gap junctions between cells,thereby increase bystander effect of HSV-tk/GCV system achieve the synergies of suicide gene therapy system on killing the hepatocarcinoma cells.更多还原