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3×10~3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。
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.
引文
[1]陈诗书,唐展云。联合免疫基因与自杀基因治疗肿瘤研究进展。中国肿瘤,1999;8(5):216-218
[2]孙春晓,何荣根.恶性肿瘤自杀基因治疗研究进展.实用肿瘤杂志,1999;14(4):255
[3]Rochlitz CF.Gene therapy of cancer.Schweiz Med Wochenschr,2001:131(1-2):1-4
[4]赵亚刚,张学庸,惠宏襄,等.逆转录病毒介导的HSV-tk/GCV对肝癌细胞的影响.中华肝脏病杂志,1999:7(2);110-113
[5]刘为池,叶钢,张荣贵.逆转录病毒介导HSV-tk基因治疗膀胱癌及旁杀伤效应的观察.重庆医学,2003:32(2);132-134
[6]Moolten FL.Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes:paradigm for a prospective cancer control strategy.Cancer Res,1986:46(10):5276-5281
[7]Denning C,Pitts JD.Bystander effects of different enzyme-prodrug systems for cancer gene therapy depend on different pathways for intercellular transfer of toxic metabolites,a factor that will govern clinical choice of appropriate regimes.Hum Gene Ther,1997,8(15):1825-1835
[8]Kumar NM,Gilula NB.The gap junction communication channel.Cell,1996,84(3):381-388.
[9]Mesnil M,Piccoli C,Tiraby G,et al.Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins.Proc Natl Acad Sci USA,1996,93(5):1831-1835
[10]Jou YS,Layhe B,Matesic DF,et al.Inhibition of gap junctional intercellular communication and malignant transformation of rat liver epithelial cells by neu oncogene.Carcinogenesis,1995:16(2):301-311
[11]毛丽敏,王勤,王菊芳,等.肿瘤细胞的间隙连接。国外医学肿瘤学分册,2003,30(4):264-266
[12]Murray SA,Davis K,Fishman LM,et al.Alphal connexin 43 gap junctions are decreased in human adrenocortical tumors.J Cln Endocrinol Metab,2000,85(2):890-895
[13]孙文海,刘华敏.李玉军,等.缝隙连接蛋白43和E钙黏附素表达与喉癌生物学行为关系的研究。中华耳鼻咽喉科杂志2004,39(5):293-297
[14]Cirenei N,Colombo BM,Mesnil M,et al.In vitro and in vivo effects of retrovirus-mediated transfer of the connexin 43 gene in malignant gliomas:consequences for HSV-tk/GCV anticancer gene therapy.Gene Ther,1998,5(9):1221-1226.
[15]Zhang LX,Acevedo P,Guo H,et al.Upregulation of gap junctional communication and connexin43 gene expression by carotenoids in dermal fibroblast but not in human keratinocytes.Mol Carcinog,1995,12(1):50-58.
[16]Shinoura N,Chen L,Wani M A,et al.Protein and messenger RNA expression of connexin43 in astrocytomas:implications in brain tumor gene therapy.J Neurosurg,1996;84(5):839-845.
[17]Shirakawa T,Gardner TA,Kosc,et al.Cytotoxicity of adenoviral-mediated cytosine deaminase plus 5-fluorocytosine gene therapy is superior to thymidine kinase plus acyclovir in a human renal cell carcinoma model.J Urol,1999;162(3Pt 1):949-954.
[18]赵良辅,严述常.张玉顺,等。六味地黄丸对诱变和自发肿瘤的抑制作用.中西医结合杂志,1990:10(7):433
[19]姜廷良.严述常,王素芬,等。六味地黄丸防治肿瘤的实验研究.中药方剂研究,1983:6:71
[20]Carystinos GD,KatabiMM,Laird DW,et al.Cyclic-AMP induction of gap junctional intercellular communication increases bystander effect in suicide gene therapy.Clin Cancer Res,1999,5(1):61-68.
[21]Check E.Gene-therapy trials to restart following cancer risk review.Nature,2005,434(7030):127.
[22]Tanaka M,Grossman HB.Connexin 26 gene therapy of human bladder cancer:induction of growth suppression,apoptosis,and synergy with Cisplatin.Hum Gene Ther.2001,12(18):2225-2236.
[23]王建立,徐克森.寿楠海.肝癌的基因治疗.中国现代普通外科进展,2004,7(3):133
[24]Zatloukal K,Schmidt W,Cotten M.Somatic gene therapy for cancer:the utility of transferrinfection in generating 'tumor vaccines'.Gene,1993;135(1-2):199-207.
[25]Kobayashi H,Takemura Y,Miyachi H.Novel approaches to reversing anti-cancer drug resistance using gene-specific therapeutics.Hum Cell,2001;14(3):172-175.
[26]贺平,叶胜龙,汤钊猷.恶性肿瘤基因治疗研究进展。中国肿瘤生物治疗杂志,1997,4(4):307
[27]Sorrentino BP,McDonagh KT,Woods D,et al.Expression of retroviral vectors containing the human multidrug resistance l cDNA in hematopoietic cells of transplanted mice.Blood,1995;86(2):491-496.
[28]蔡洪培,邓志华,李石等.反义核酸抗肿瘤治疗研究中靶基因的选择.中国肿瘤生物治疗杂志,1998。5(2):148-151.
[29]Nguyen TH,OBerholzer J,Birraux J,et al.Highly efficient lentiviral vector-mediated transduction of nondividing,fully reimplantable primary hepatocytes.Mol Ther.2002,6(2):199-209.
[30]Kang Y,Stein CS,Heth JA,et al.In vivo gene transfer using a nonprimate lentiviral vector pseudotyped with Ross River Virus glycoproteins.J Virol.2002,76(18):9378-9388.
[31]孙春晓,何荣根。恶性肿瘤自杀基因治疗研究进展.实用肿瘤杂志,1999,14(4):255.
[32]Germano IM,Fable J,Gnhekin SH,et al.Adenovirus/herpes simplex-thymidine kinase/ganciclovir complex:preliminary results of a phase I trial in patients with recurrent malignant gliomas.J Neurooncol,2003,65(3):279-289.
[33]Prados MD,McDermott M,Chang SM,et al.Treatment of progressive or recurrent glioblastoma multiforme in adults with herpes simplex virus thymidine kinase gene vector-producer cells followed by intravenous ganciclovir administration:a phase Ⅰ/Ⅱ multi-institutional trial.J Neurooncol,2003,65(3):269-278.
[34]Moolten FL.Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes:paradigm for a prospective cancer control strategy.Cancer Res,1986,46(10):5276-5281.
[35]Culver KW,Ram Z,Wallbridge S,et al.In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors.Science,1992,256(5063):1550-1552.
[36]李祷．HSV-tk基因／GCV系统治疗脑胶质瘤的研究进展．肿瘤防治研究,1998,25(6)：500-502．
[37]Freeman SM,Abboud CN,Whartenby KA,et al.The “bystander effect”:tumorregression when a fraction of the tumor mass is genetically modified.Cancer Res,1993,53(21):5274.
[38]Consalvo M,Mullen CA,Modesti A,et al.5-Fluorocytosine-induced eradication of murine adenocarcinomas engineered to express the cytosine deaminase suicide gene requires host immune competence and leaves an efficient memory.J Immunol.1995,154(10):5302-5312.
[39]Caruso M,Panis Y,Gagandeep S,et al.Regression of established macroscopic liver metastases after in situ transduction of a suicide gene.Proc Natl Acad Sci USA,1993,90(15):7024-7028.
[40]Gagandeep S,Brew R,Green B,et al.Prodrug-activated gene therapy:involvement of an immunological component in the “bystander effect”.Cancer Gene Ther,1996,3(2):83-88
[41]Hogge GS,Burkholder JK,Culp J,et al.Preclinical development of human granulocyte-macrophage colony-stimulating factor-transfected melanoma cell vaccine using established canine cell lines and normal dogs.Cancer Gene Ther,1999,6(1):26-36.
[42]Shi FS,Weber S,Gan J,et al.Granulocyte-macrophage colony-stimulating factor(GM-CSF)secreted by cDNA-transfected tumor cells induces a more potent antitumor response than exogenous GM-CSF.Cancer Gene Ther.1999,6(1):81-88.
[43]Dilber MS,Abedi MR,Bjorkstrand B,et al.Suicide gene therapy for plasma cell tumors.Blood,1996,88(6):2192-2196.
[44]Hirschowitz EA,Ohwada A,Pascal WR,et al.In vivo adenovirus-mediated gene transfer of the Escherichia coli cytosine deaminase gene to human colon carcinoma-derived tumors induces chemosensitivity to 5-fluorocytosine.Hum Gene Ther,1995,6(8):1055-1058.
[45]Ram Z,Walbridge S,Shawker T,et al.The effect of thymidine kinase transduction and ganciclovir therapy on tumor vasculature and growth of 9L gliomas in rats.Neurosurg,1994,81(2):256—260
[46]Vrionis FD.Wu JK,Qi P,et al.The bystander effect exerted by tumor cells expressing the herpes simplex virus thy midine kinase(HSV-tk)gene is dependent on connexin expression and cell Communication via gap junction.Gene Ther,1997,4(6):577-585.
[47]McMasters RA.Saylors RL,Jones KE,et al.Lack of bystander killing in herpes simplex virus thymidine kinase-transduced colon cell lines due to deficient connexin43 gap junction formation.Hum Gene Ther,1998,9(15):2253-2261.
[48]Vrionis FD,Wu JK,QI PI,et al.The bystander effect exerted by tumor cells expressing the herpes simplex virus thymidine kinase(HSVtk)gene is dependent on connexin expression and cell communication via gap junctions.Gene Ther,1997,4(6):57-585.
[49]Mesnil M,Piccoli C,Tiraby G,et al.Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins.Proc Natl Acad Sci USA,1996,93(5):1831-1835.
[50]Duflot-Dancer A,Piccoli C,Rolland A,et al.Long-term connexin-mediated bystander effect in highly tumorigenic human cells in vivo in herpes simplex virus thymidine kinase/ganciclovir gene therapy.Gene Ther,1998,5:1372-1378
[51]Park JY,Elshima AA,Amin K,et al.Retinoids augment the bystander effect in vitro and in vivo in herpes simplex virus thymidine kinase/ganciclovir-mediated gene therapy.Gene Ther,1997,4(9):909-917.
[52]Carystinos GD,Katabi MM,Laird DW,et al.Cyclic-AMP induction of gap junctional intercellular communication increases bystander effect in suicide gene therapy.Clin Cancer Res,1999,5(1):61～68.
[53]Touraine RL,Ishii-Morita H,Ramsey J,et al.The bystander effect in the HSVtk/ganciclovir system and its relationship to gap junctional communication.Gene Ther,1998,5(12):1705-1711.
[54]Touraine RL,Vahanian N,Ramsey WJ,et al.Enhancement of the herpes simplex virus thymidine kinase/ganciclovir bystander effect and its antitumor efficacy in vivo by pharmacologic manipulation of gap junctions.Hum Gene Ther,1998,9(16):2385-2391.
[55]Mesnil M,Piccoli C,Yamasaki H.A tumor suppressor gene,Cx26,also mediates the bystander effect in HeLa cells.Cancer Res,1997,57(14):2929-2932.
[56]Colombo BM,Benedetti S,Ottolenghi S,et al.The “bystander effect”:association of U-87 cell death with ganciclovir-mediatedapoptosis of nearby cells and lack of effect in athymic mice.Hum Gene Ther,1995,6(6):763-772.
[57]Zhang LX,Acevedo P,Guo H,et al.Upregulation of gap junctional communication and connexin43 gene expression by carotenoids in human dermal fibroblasts but not in human keratinocytes.Mol Carcinog,1995,12(1):50—58.
[58]Carystinos GD,Katabi MM,Laird DW,et al.Cyclic-AMP induction of gap junctional intercellular communication increases bystander effect in suicide gene therapy.Clin Cancer Res,1999,5(1):61-68.
[59]Jansen LA,Mesnil M,Jongen WM.Inhibition of gap junctional intercellular communication and delocalization of the cell adhesion molecule E-cadherin by tumor promoters.Carcinogenesis,1996,17(7):1527-1531.
[60]Cirenei N,Colombo BM,Mesnil M,et al.In vitro and in vivo effects of retrovirus-mediated transfer of the connexin43 gene in malignant gliomas:consequences for HSV-tk/GCV anticancer gene therapy.Gene Ther,1998,5(9):1221-1226.
[61]Landesman Y,White TW,Starich TA,et al.Innexin-3 forms connexin-like intercellular channels.J Cell Sci,1999,112(ptl4):2391-2396.
[62]Moolten FL,Wells JM,Heyman RA,et al.Lymphoma regression induced by ganciclovir in mice bearing a herpes thymidine kinase transgene.Hum Gene Ther,1990,1(2):125-134.
[63]Sullivan R,Ruangvoravat C,Joo D,et al.Structure,sequence and expression of the mouse Cx43 gene encoding connexin43.Gene.1993,130(2):191-199.
[64]瞿中和,王喜忠,丁明零,等．细胞生物学．北京：高等教育出版社,2003：124-125．
[65]Jeong SH,Cho MH,Cho JH.Effects of cadmium on gap junctional intercellular communication in WB-F 344 rat liver epithelial cells.Human & Experimental Toxicology,2001,20(11):577-583.
[66]Furshpan EJ,potter DD,Transmission,et al.The giant motorsynapses of the crayfish.J Physiol,1959,145:289-325.
[67]Revel JP,Karnovsky MJ.Hexagonal array of subunits in intercellular junctions of the mouse hearts and liver.J Cell Biol,1967,33(3):7212.
[68]Makowski L,Caspar DL,Phillips WC,etal.Gap junction structures.Ⅵ Variation and conservation in connexon conformation and packing.Biophys J,1984,45(1):208-218.
[69]Leithe E,Cruciani V,Sanner T,et al.Recovery of gap junctional intercellular communication after phorbol ester treatment requires proteasomal degradation of protein kinase C.Carci nogenesis,2003,24(7):1239-1245.
[70]Beyer EC,Paul DL,Goodenough DA.Connexin family of gap junction proteins.J Membr Biol,1990,16:187-194.
[71]Cottrel GT,Burr JM.Functional consequences of heterogeneousgap junction channel formation and its influence in health and disease.Biochim Biophys Acta.2005,1711(2):126-141.
[72]Lars-Oliver K,Pauline P,Niloofar AA,et al.2-Methyl-l,4-Naphthoquinone,vitamin K3,decreases gap-junctional intercellular communication via activation of the epidermal growth factor receptor /extracellular signal-regulated kinase cascade.Cancer Res,2002,62(17):4922-8.
[73]Nagy JI,Rash JE.Connexins and gap junctions of astro cytes and oligodendro cytes in the CNS.Brain Res Brain Res Rev,2000,32(1):29-44.
[74]Gong X,Agopian K,Kumar NM,et al.Genetic factors influence cataract formation in alpha 3 connexin knockout mice.DevGenet,1999,24(1-2):27-32.
[75]White TW,Goodenough DA,Paul DL.Targeted ablation of connexin 50 in mice results in microphthalmia and zonular pulverulent cataracts.J Cell Biol,1998,43(3):815-825.
[76]施正专,沈守荣,邹益友,等.Cx基因在胃癌细胞株中的表达及诱导分析.癌症,2000.19(5):423-425。
[77]Gabriel HD,Jung D,Butzler C,et al.Transplacental uptake of glucose is decreased in embryonic lethal connexin 26 deficient mice.J Cell Biol,1998,140(6):1453-1461.
[78]Scott DA,Kraft ML,Stone EM,et al.Connexinmutationsand hearing loss.Nature,1998,391(6662):32.
[79]Scherer SS,Xu YT,Nelles E,et al.Connexin 32-null mice develop demyelinating peripheral neuropathy.Glia,1998,24(1):8-20.
[80]Scherer SS,Xu YT,Nelles E,et al.Connexin 32-null mice develop demyelinating peripheral neuropathy.Glia,1998,24(1):8-20.
[81]Tsuda H,Asamoto M,Baba H,et al.Cell proliferation and advancement of hepatocarcinogenensisin the rat are associated with a decrease in connexin 32expression.Carcinogenesis,1995,16(1):101-105.
[82]Wilecke K,Eiberger J,Degen J,et al.Structural and functionaldiversity of connexin genes in the mouse and human genome.8iol Chem,2002,383(3):725-737.
[83]Niloofar AA,Wilhelm S,Helmut S.(-)-Epicatechin effects in rat liver epithelial cells:Stimulation of gap junctional communication and counteraction of its loss due to the tumor promoter 12-Otetradecanoylthorbol-13-acetate.Biochem Pharmacol,2002,63(12):2145-9.
[84]Vinken M,Vanhaecke T,Papeleu P,et al.Connexins and theirchannels in cell growth and cell death.Cell Signal,2006,18(5):592-600.
[85]Chipman JK,Mally A,Edwards GO.Disruption of gap junctionsin toxicity and careinogenicity.Toxicol Sci,2003,71(2):146-153.
[86]Fujimoto E,Yano T,Sato H,et al.Cytotoxic efect ofthe Her-2/Her-1 inhibitor PKI-166 on renal cancer cells expressing the connexin 32 gene.J Pharmaeol Sei,2005,97(2):294-298.
[87]Huang RP,Hossain MZ,Huang R,et al.Connexin 43(cx43) enhances chemotherapyinduced apoptosis in human glioblastomaeel Is.lnt J Cancer,2001,92(1):130-138.
[88]Hechki MP,Toti RM,Femstrom MJ,et al.Liver cell-specific transeriptional regulation of connexin32.Biochim Biophys Acta,2000,1491(1-3):107-122.
[89]Teunissen BE,Bierhuizen MF.Transcriptional control of myocardial connexins.Cardiovase Res,2004,62(2):246-255.
[90]Chipman JK,Mally A,Edwards GO.Disruption of gap junctionsin toxicity and careinogenicity.Toxicol Sci,2003,71(2):146-153.
[91]Lampe PD,Lau AF.The effects of connexin phosphorylation on gap junctional communication.Int J Biochem Cell Biol,2004,36(7):1171-1186.
[92]Elcock FJ,Deag E,Roberts RA,et al.Nafenopin causes proteinkinase C-mediated serine phosphorylation and loss of funotion of connexin 32 protein in rat hepatocytes without aberrant expression or localization.Toxicol Sci,2000,56(1):86-94.
[93]Yinken M,Vanhaecke T,Papeleu P,et al.Connexins and their channels in cell growth and cell death.Cell Signal,2006,18(5):592-600.
[94]Hernandez-Blazquez FJ,Joazeiro PP,Omori Y,et al.Control of intracellular movement of connexins by E-cadherin in murine skin papilomacels.Exp Cell Res,2001,270(2):235-247.
[95]Han LQ,Gao J,Dong HY.Studies on the gap junctional intercellular communication of human nasopharyngeal carcinoma cells and effect of RII.Chung Hua Chung Liu Tsa Chih,1994,16(5):345-8.
[96]Zhang ZQ,Hu Y,Wang BJ,et al.Effective asymmetry in gap junctional intercellular communication between populations of human normal lung fibroblasts and lung carcinoma cells.Carcinogenesis,2004,25:473-482.
[97]Margaret Ⅱ,James E.A fluoresecence phorobleaching assay of gap junction medicated communication between human cells.Science,1983,232:525.
[98]Chaudnuri R.Gap junction intercellular communication in mouse lung epithelial cell line:effects of cell transformation and tumor promoters.Cancer Lett,1993,71(1-3):11.
[99]Martin PE,Blundell G,Ahmad S,et al.Multiple pathways in the trafficking and assembly of connexin 26,32 and 43 into gap junction intercellular communication channels.Cell Sci,2001,114:3845-55.
[100]周逢海,宋波,金锡御.激光扫描共聚焦显微镜在细胞间隙连接研究中的应用.细胞生物学杂志,2003,25(5):283-284.
[101]Carruba G,Webber MM,Bello-Deocampo D,et al.Laser scanning analysis of cell-cell communication in cultured human prostate tumor cells.Anal Quant Cytol Histol,1999,21(1):54-58.
[102]马向东,王文亮,徐力青,等.肝癌细胞间隙连接蛋白Cx32,Cx43的流式细胞仪分析.细胞分子与免疫学杂志,1999,15(1):51.
[103]孙华,刘耕陶。细胞间隙连接通讯与肿瘤.中国药理学通报.2004,20(11):1205-1208.
[104]刑毅飞,肖亚军,鲁功成.连接蛋白介导的GJIC与肿瘤自杀基因治疗时旁观者效应的关系.国外医学.生理、理科学与临床分册.2001,21(4):246-248.
[105]王卫东综述,陈正堂,王志新审阅.缝隙连接与自杀基因旁观者效应研究进展.中国肿瘤生物治疗杂志.2001,8(3):231-233.
[106]袁慧,浦跃朴.细胞间隙连接通讯与癌变关系的研究进展.中国预防医学杂志.2004,15(11):67-68.
[107]姚庆华,郭勇.细胞间隙连接通讯、连接蛋白与肿瘤的抑制.肿瘤研究与临床。2005,17(1):67-70.
[108]Omori Y,Zaidan Dagli ML,Yamakage K,et al.Involvement of gap junctions in tumor suppression:analysis of genetically-manipulated mice.Mutat Res,2001,477(1-2):191-196.
[109]毛丽敏,王勤,王菊芳.肿瘤细胞的间隙连接.国外医学肿瘤学分册.2003,30(4):266-269.
[110]Kwak BR,van Veen TA,Analbers LJ,et al.TPA increases conductance by decreases permeability in neonatal rat cardiomyocyte gap junction channels.Exp Cell Res,1995,220(2):456-463.
[111]King TJ,Lampe PD.Altered tumor biology and tumorigenesis in irradiated and chemical carcinogen-treated single and combined connexin32/p27Kipl-deficient mice.2005,12(5-6):293-305.
[112]Ale Agha N,Stahl W,Sies H.(-)-Epicatechin effects in rat liver epithelial cells:stimulation of gap junctional communication and counteraction of its loss due to the tumor promoter 12-0-tetradecanoylphorbol-13-acetate.Biochem Pharmacol.2002,63(12):2145-2149.
[113]Edward Leithe,Edgar Rivedal.Ubiquitination and Down-regulation of Gap Junction Protein Connexin-43 in Response to 12-0-Tetradecanoylphorbol 13-Acetate Treatment.THE JOURNAL OF BIOLOGICAL CHEMISTRY.2004,279(48):50089-50096.
[114]Roberts PC,Mottillo EP,BaxaAC,et al.Sequential molecular and cellular events during neoplastic progression:a mouse syngeneic ovarian cancer model.Neoplasia.2005 Oct,7(10):944-956.
[115]Yamssaki H,Omori Y,Zaidan--Dagli ML,et al.Genetic and epige--netic changes Of interceluar communication genes during multistagecinogenesis.Cancer Detect Prev,1999,23:273-279.
[116]Trosko JE.The role Of stem cells and gap junctional intercellular communication in carcinogenesis.J Biochem Mol Bid.2003,36:43-48.
[117]Plante I,Charbonneau M,CyrD G.Decreased gap junctional intercellular communication in hexachlorobenzene induced gender specific hepatic tumor formation in rat.Carcinogenesis,2002,23:1243-1249.
[118]Lee SW,Tomasetto C,Paul D,et al.Transcriptional down regulation of gap junction proteins blocks functional communication in human manmary tumor cell lines.J cell Biol.1992,118:1213-1221.
[119]Mesnil M,Krutovskikh V,Piccoli C,et al.Negative growth control of HeLa cells by connexin genes:connexin.Cancer Res,1995.55(3):629-639.
[120]Hirschi KK,Xu CE,Tsukamoto T,et al.Gap junction genes Cx26 and Cx43individually suppress the cancer phenotypes of human mammary carcinoma cells and restore differentiation potential.Cell Growth Differ,1996,7(7):861-870.
[121]Moennikea O,Buchmann A,Ou T,et al.The effect of Connexin 32 null mutation on hepatocarcinogenesis in different mouse strains.Carcinogenesis,1999,20:1379-1382.
[122]Grossman HB,Liebert M,Lee IW.Decreased connexin expression and intercellular communication in human bladder cancer cells.Cancer Res,1994,54(11):3062-3065.
[123]Naus CC,Elisevich K,Zhu D,et al.In vivo growth of C6 glioma cells transfected with connexin43 cDNA.Cancer Res,1992,52(15):4208-4213.
[124]Shinoura N,ChenL,Wani MA,et al.Protein and messenger RNA expression of connexin43 in astrocytomas:implications in brain tumor gene therapy.J Neurosurg,1996,84(5):839-845.
[125]林仲翔,张志谦,王耐勤.间隙连接基因Cx43表达对肺癌细胞体内成瘤生长的抑制.中华肿瘤杂志,1997,19(4):253-255.
[126]夏之柏,浦佩玉,王广秀.转染连接蛋白基因对胶质瘤细胞细胞间隙连接通讯及其生长变化的研究.中华实验外科杂志,2003,7:161-167.
[127]Mesnil M,Yamasaki H.Bystander effect in herpes simplex virus-thymidine kinase/ ganciclvir cancer gene therapy:role of gap-junctional intercellular communication.Cancer Res,2000,60(15):3989-3992.
[128]Tsudan H,Asamoto M,Baba H,et al.Cell proliferation and advancement of hepatocarcinogenesis in the rat are associated with a decrease in connexin 32expression.Carcinogenesis,1995,16:101.
[129]Tanaka M,Grossman HB.Connexin 26 gene therapy of human bladder cancer;induction of growth suppression,apoptosis,and synergy with cisplation.Hum gene Ther,2001,12:2225-2236.
[130]Carrio M,Mazo A,Lopez-Iglesias C,et al.Retrovirus-mediated transfer of the herpes simplex virus thymidine kinase and connexin26 genes in pancreatic cells results in variable efficiency on the bystander killing:implications for gene therapy.Int J Cancer,2001,94(1):81-88
[131]马向东,隋延仿,王文亮.间隙连接蛋白Cx32在肝细胞癌组织中的表达.第四军医大学学报,1998:50
[132]Kumar NM,Gilula NB.The gap junction communication channel.Cell,1996,84(2):381-388.
[133]陈孝平,张必翔.我国原发性肝癌治疗的现状及评价.中华肝脏病杂志,2001,9(3):132
[134]Qian C,Bilbao R,Bruna O,et al.Induction of sensitivity to ganciclovir in human hepatocellular carcinoma cells by adenovirus- mediated gene transfer of herpes simplex virus thymidine kinase.Hepatology,1995,22(1):118-123.
[135]Kwon HC,Kim JH,Kim KC,et al.In vivo antitumor effect of herpes simplex virus thymidine kinase gene therapy in rat hepatocellular carcinoma:feasibility of adenovirus-mediated intra-arterial gene delivery.Mol Cells,2001,11(2):170-178
[136]Kuriyama S,Mitoro A,Yamazaki M,et al.Comparison of gene therapy with the simplex virus thymidine kinase gene and the bacterial cytosine deaminase gene for the treatment of hepatocellular carcinoma,Scand J Gastroenterol,1999,34(10):1033-1041
[137]Sung MW,Yeh HC,Thung SN,et al.Intratumoral adenovirus mediated suicide gene transfer for hepatic metastases from colorectal adenocarcinoma:results of a phase I clinical trial,Mol Ther,2001,4(3):182-191
[138]郁仁存,姜廷良,于尔辛主编。肿瘤研究.上海科学技术出版社,1991:17.
[139]齐春会。张永祥,李凤仙.等.六味地黄多糖体外抗肿瘤作用的初步研究.中国药理学通报.1999,15(4):322
[140]赵凤鸣,王明艳,吴海涛等.两首滋阴方药对环磷酰胺诱发的微核的抑制作用.福建中医药,1998,29(4):23
[141]许继平,周振鹤,杨锋,等.六味地黄口服液抗肿瘤化疗药物毒副作用的研究.中国中西医结合杂志.1992,12(12):734-737
[142]贾振和,等.六味地黄丸预防食道癌放疗中外周血象降低54例.中医杂志,2001,42(7):402.
[143]王秀荣,张永红,王秀英,王辉.六味地黄汤对糖皮质激素肾阴虚模型免疫功能的影响.河北医科大学学报,2005,26(6):451-452
[144]姜延良.六味地黄汤防治肿瘤的实验研究,中医杂志,1983:24(6):71
[145]曲靖.六味地黄丸(汤)的现代药理研究进展.湖南中医杂志.2007,23(6):78-79
[146]张永祥.六味地黄汤现代药理学及化学的初步研究。基础医学与临床,2000,20(5):15-18
[147]尚西亮,李学东,傅华群.缝隙连接细胞间通讯在炎症、损伤修复和疾病中的作用.国外医学(老年医学分册).2006,27(3):137-140
[148]谭宇蕙,吴映雅,杜标炎,等.大鼠肝癌细胞HSV-tk/GCV自杀基因系统的构建及其旁观者效应.世界华人消化杂志,2005.13(17);206
[149]杜标炎,张小贺,谭宇蕙,等.六味地黄丸含药血清调控黑色素瘤B16细胞株缝隙连接蛋白表达的作用。广州中医药大学学报.2009,26(2):152-156
[150]杜标炎,张爱娟,谭宇蕙,等.六味地黄丸对自杀基因系统杀伤肝癌细胞增效作用的量效关系。广州中医药大学学报,2009,26(1):59-61
[151]杜标炎,张爱娟,谭宇蕙,等.自杀基因系统联合六味地黄丸对肝癌细胞杀伤的协同作用.广州中医药大学学报,2008,25(4):319-324
[152]杜标炎,王慧峰,谭宇蕙,等.六味地黄丸对小鼠移植性肝癌自杀基因治疗的增效作用的病理学研究。广州中医药大学学报,2007,24(5):386-391
[153]杜标炎,王慧峰,谭宇蕙,等.六味地黄丸对小鼠移植性肝癌自杀基因治疗的增效作用。广州中医药大学学报,2007,24(2):132-137
[154]#12
[155]崔晓兰,贺玉琢.高英杰中药药理研究的新思路-中药血清药理学.中国中医药科技1997.4(4):239-250.
[156]荣一纯,丁霞,贲长恩,等.中药血清药理学应用展望.北京中医药大学学报.1999。22(4):42-44.
[157]赵万红,曹永孝,袁泽飞.中药血清药理学的方法探讨.中药新药与临床药理,2002,13(2):122-125.
[158]蒙一纯,丁霞,郭顺根。中药血清药理学应用研究展望。北京中医药大学学报,1999,22(4):42-45.
[159]周明眉,扬奎,姜远平。等.中药血清药理学的方法学研究-含药血清低温保存和血清灭活的影响。中药药理与临床。1999,15(2):44-47.
[160]杨奎.含黄芩血清及黄芩甙影响内生致热原产生的研究.中药药理与临床,1994,10(6):13.
[161]杨奎.含香薷、羌活胜湿汤和九味羌活丸血清对内生致热原产生的影响.中药药理与临床,1995,9(4)
[162]杨振江.髓清丸对HEL细胞系作用的血清药理学研究。中国中医基础医学杂志,2003,9(6):30-32.
[163]邱幸生.益气通络方对缺氧再给氧心肌细胞bcl-2基因表达影响的血清药理学研究.中国中医基础医学杂志,2001,7(3):38-40.
[164]梁春敏.玉屏风散对小鼠免疫调节作用的血清药理学研究.上海免疫学杂志,2003,23(6):385-388.
[165]杨奎.中药血清药理学研究方法在主要抗菌实验中的应用.中药药理与临床,1995,11(特):121.18.
[166]韩立民,刘波,徐彭.淫羊藿对成骨细胞增殖的血清药理学研究.中医药学刊,2003,21(5):678-680.
[167]李翠屏.益气通络方对大鼠皮层神经元缺氧凋亡影响的血清药理学研究.中药药理与临床,2002,18(5):39-41.
[168]刘成海.扶正化瘀方药物血清对大鼠肝贮脂细胞增殖及胶原合成的影响.中国实验方剂学杂志,1996,2(2):16.
[169]夏誉薇。女贞孕育汤对鸡胚绒毛尿囊膜血管生成模型血清药理学研究.中医药学刊,2003,21(4):531-534.
[170]Mesnil M,Yamasaki H.Bystander effect in herpes simplex virus-thymidine kinase ganciclovir cancer gene therapy:role of gap-unctional intercellular communication.Cancer Res,2000,60:3989-3999.
[171]Juan C,S AEZ,VIVIANA M,BERTHOUD,et al.Plasma Membrane Channels Formed by Connexins:Their Regulation and Functions.Physiol Rev.2003,83:1360-1400
[172]Mesnil M,Piccoli C,Tiraby G,et al.Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins.Proc Natl Acad Sci U S A,1996,93(5):1831.
[173]Jordan K,Chodock R,Hand AR,et al.The origin of annular junctions:a mechanism of gap junction internalization.J Cell Sci,2001,114(4):763-773.
[174]Bi WL,Parysek LM,Warnick R,et al.In vitro evidence that metabolic cooperation is responsible for the bystander effect observed with HSV tk retroviral gene therapy.Human Gene Therapy,1993,4(6):725.
[175]Freeman SM,Abboud CN,Whartenby KA,et al.The "bystander effect":tumorregression when a fraction of the tumor mass is genetically modified.Cancer Res,1993,53(21):5274.
[176]Mohr L.Hepatocellular carcinoma:novel therapeutic approaches.Schweiz Rundsch Med Prax,2007,96(14):553.
[177]Lemken ML,Graepler F,Wolf C,et al.Fusion of HSV-1 VP22 to a bifunctional chimeric SuperCD suicide gene compensates for low suicide gene transduction efficiencies.Int J Oncol,2007,30(5):1153.
[178]孙春晓,何荣根.恶性肿瘤自杀基因治疗研究进展.实用肿瘤杂志,1999:14(4):255。
[179]杜标炎,徐勤,吴绍锋,等.六味地黄汤对糖皮质激素肾阴虚模型免疫器官淋巴细胞凋亡的抑制作用(Ⅰ).广州中医药大学学报,2000,17(3):204.
[180]刘福君,茹祥斌。地黄及六味地黄汤(丸)的免疫药理及抗肿瘤作用。中草药,1996,27(2):116.
[181]宋丽丽,张瑜,张大禄。六味地黄方的免疫、抗肿瘤药理研究.中成药,2001,23(12):910
[182]杜标炎,王慧峰,谭宇蕙,等.六味地黄丸对小鼠移植性肝癌自杀基因治疗的增效作用。广州中医药大学学报,2007,24(2):132。
[183]杜标炎,张爱娟.谭宇蕙,等.自杀基因系统联合六味地黄丸对肝癌细胞杀伤的协同作用.广州中医药大学学报,2008,25(4):319.
[184]杜标炎,谭宇蕙,吴映雅,等.逆转录病毒载体介导HSV1-tk/GCV肿瘤自杀基因治疗系统的构建。广州中医药大学学报,2004,21(5):395.
[185]刘为池,叶刚.自杀基因治疗旁观者效应研究进展.国外医学:泌尿系统分册.2000,20(增刊):121.
[186]张杰,张红英,步宏,等.不同转移潜能的人体横纹肌肉瘤细胞系细胞间通讯的研究.中华病理学杂志.2001,30(6):48--451.
[157]Carruba G,Webber MM,Bello-Deocampo D,et al.Laser scanning analysis of cell-cell communication in cultured human prostate tumor cells.Anal Ouant Cytol Histol,1999,21(1):54-58.
[188]周逢海,宋波,金锡御,等.荧光光漂白恢复技术在检测膀胱平滑肌细胞间通讯功能中的应用.第三军医大学学报,2003,25(24):2208-2211。
[189]徐营,雷蕾,刘忠华等.应用激光扫描共聚焦显微镜FRAP技术研究兔早期胚胎发育中细胞间隙连接介导通讯。细胞生物学通讯,2002,24(5):310-312.
[190]Grossman HB,Liebert M,Lee IW.Decreased connexin expression and intercellular communication in human bladder cancer cells.Cancer Res,1994,54(11):3062-3065.
[191]Shinoura N,Chen L,Wani M A,et al.Protein and messenger RNA expression of connexin 43 in astrocytomas:implications in brain tumor gene therapy.J Neurosurg,1996:84(5):839-845.
[192]Clairmmont A,Sies H.Evidence for a posttranscriptional effect of retinoic acid on connexin 43 gene expression via the 3'-untranslated region.FEBS Lett,1997,419:268.
[193]Lars-Oliver K,Pauline P,Niloofar AA,et al.2-Methy1-1,4-Naphthoquinone,vitamin K3,decreases gap-junctional intercellular communication via activation of the epidermal growth factor receptor/extracellular signal-regulated kinase cascade.Cancer Res,2002,62(17):4922-8.