HIV Vpr蛋白应用于特异性肝癌基因治疗的研究
摘要
原发性肝癌（primary carcinoma of liver）是指自肝细胞或胆管
细胞发生的癌。原发性肝癌是世界上恶性程度极高,且预后极差的恶性肿
瘤之一,每年全球约有 25 万人患病。在我国肝癌目前已处于恶性肿瘤死
亡率的第二位,每年至少有 10 万名新发现病人，11 万名肝癌病人死亡，
占全球肝癌死亡数的 45%。
尽管目前肝癌的治疗方法有许多种，但治疗效果都十分有限。大部
分肝癌病人对化疗和放疗均不敏感，因此寻找新的有效的治疗方式一直
是研究者追求的目标。随着分子生物学和免疫学理论及技术的不断发展
和对肿瘤发病机制认识的不断深入，利用基因工程手段对肝癌进行基因
治疗已取得了很大成功，日益受到研究者的重视。基因治疗及有可能成
为一种新的治疗方式应用于肿瘤的治疗之中。
腺病毒载体因其具有安全性好、外源基因表达效率高、滴度高、既可感
染分裂期细胞又可转染非分裂期细胞以及包装容量大等优点而成为当今
使用最多的病毒载体之一。
HIV vpr 基因是 I 型人类免疫缺陷病毒的非结构基因，编码一 14Kda
大小分子量的蛋白。研究表明，该蛋白是 HIV 重要的调控蛋白，Vpr 蛋
白不依赖于 HIV 其他蛋白的存在即可引起细胞的 G2 周期阻滞和诱导细
胞凋亡，单独表达的 Vpr 蛋白也可引起细胞的 G2 周期阻滞和诱导细胞凋
亡。文献报道 Vpr 蛋白可引起分裂细胞，包括肿瘤细胞发生凋亡。我们
1
的前期工作表明，HIV Vpr 对 Hela 细胞表现出明显的细胞周期阻滞作用。
由此可见 Vpr 很有可能作为一种新的治疗性基因而应用于以抑制肿瘤生
长为目的的基因治疗中。
目前在多数原发性肝细胞中有甲胎蛋白的表达，将外源治疗基因置
于 AFP 特异性启动子增强子下游，通过肝癌中 AFP 的特异性反式作用于
该启动子，以激活转录，可实现目的基因的选择性表达，从而可实现有
效的靶向性治疗，目前 AFP 启动子介导的肝癌特异性基因治疗已成为肝
癌特异性基因治疗领域的主要方面。
理想的基因治疗应具有目的基因高效性、靶向性转移和可控性表达
的特点。鉴于以上认识，我们应用非复制型腺病毒载体制备了甲胎蛋白
启动子控制下表达 HIV vpr 基因的重组腺病毒 rvAdAFP-vpr，实现其对肝
癌的特异性基因治疗作用，并对其生物学特征及其体外体内的治疗效果
进行研究，得到如下实验结果：
1．通过重组非复制型 5 型腺病毒 pAdEasy 系统，利用 E.coliBJ5183
的同源重组机制获得的甲胎蛋白启动子控制下表达 HIV vpr 基因重组非
复制型腺病毒 rvAdAFP-vpr，经 PCR 及 Southern blot 检测证实在重组腺
病毒基因组中整合有特异性的 HIV vpr 和 AFP 的上游调控序列。
2．重组腺病毒 rvAdAFP-Vpr 感染两种肝癌细胞：AFP 蛋白表达阳性
的 BEL-7402 细胞和 AFP 蛋白表达阴性的 SMMC-7721 细胞，实验结果表
明：Vpr 蛋白在 AFP 蛋白表达阳性的肝癌细胞 BEL-7402 细胞中特异性表
达，并且可引起 BEL-7402 细胞的细胞周期 G2 阻滞，G2 期细胞数目在病
毒感染后的 96 小时达到最大值，表现出一定的时间依赖性。对于 AFP
蛋白表达阴性的 SMMC-7721 细胞而言，却未能检测出 Vpr 蛋白的表达也
未见细胞周期阻滞作用。
3．通过荧光染色和和细胞线粒体膜电位改变等指标看到，重组腺病
2
毒 rvAdAFP-vpr 可诱导肝癌细胞凋亡。流式细胞术检测各个与细胞凋亡
有关的蛋白 Bax、Caspase 9、Caspase 8、Capase 3、细胞色素 C 表达情况。
结果显示，Bax 蛋白表达量明显升高，Caspase 9、Capase 3、细胞色素 C
等表达较对照都升高；而 caspase 8 蛋白表达未见明显变化。提示 Vpr 蛋
白可能通过线粒体途径引起细胞凋亡。
4．我们在裸鼠建立 AFP 蛋白表达阳性的人肝癌模型，并进行了 1
个月的分组治疗。免疫组化检测表明，rvAdAFP-vpr 可在肝癌组织中有效
表达 Vpr 蛋白。治疗效果表明，CPA 治疗组、rvAdAFP-vpr 治疗组和
rvAdAFP-vpr 联合 CPA 治疗组相对于空白对照组，都在不同程度上显著
抑制肿瘤生长，特别是 rvAdAFP-vpr 联合 CPA 组抑制肿瘤增长的作用更
为明显。CPA 治疗组、rvAdAFP-vpr 治疗组和 rvAdAFP-vpr 联合 CPA 治
疗组的抑瘤率分别达到了 41%、31%、66.7%，表明单独注射 rvAdAFP-vpr
组的抑瘤效果要好于 CPA 治疗组，说明单独注射 rvAdAFP-Vpr 即可一定
程度上起到抑制肿瘤生长的作用；另一方面，两者联合治疗组的抑瘤率
显著高于 rvAdAFP-vpr 组和 CPA 组的单独治疗，提示两者具有协同作用。
5．空白对照组和 rvAd-null 对照组的 Ki-67 指数明显高于 CPA 治疗
组、rvAdAFP-vpr 治疗组、rvAdAFP-vpr 联合 CPA 治疗组。其中对照组
和 rvAd-null 组的 Ki-67 指数对比于 rvAdAFP-vpr 联合 CPA 组具有统计学
意义，P<0.05。表明 Vpr 蛋白细胞周期 G2 期阻滞的作用有效地减缓了细
胞分裂增殖速度、抑制了肿瘤的快速增殖。CPA 治疗组、rvAdAFP-vpr
治疗组、rvAdAFP-vpr 联合 CPA 治疗组的凋亡指数明显高于对照组和
rvAd-null 组，其中 rvAdAFP-vpr 联合 CPA 组与对照组对比具有统计学意
义，P<0.05。体内实验?
Hepatomacellular carcimoma (HCC) is one of the most common malignancies
worldwide with an extremely poor prognosis. The major etiologic risk factors for HCC
development include toxin, hepatitis B virus (HBV) and hepatitis C virus(HCV) infection
as well as various inherited metabolic disorders. Surgical treatment is the most important
treatment of HCC. However, clinical observation have shown that tumor recurrence rates
are very high in patients with HCC who receive medical or surgical treatment. Thus new
treatment modalities must be pursued. With the expectation of increasing therapeutic
efficacy, gene therapy has been introduced as a new direction in treatment for HCC. Gene
therapy strategies against HCC include suppressor gene therapy, anti-sene gene therapy,
immunogene therapy, suicide gene therapy and combinanted-gene therapy. However, the
overall results of the studies are still disappointing. How to search for tumor-specific
agent of gene therapy? How to achieve appropriate spatial and temporal control of the
expression of the therapeutic gene? How to construct tumor tissue-targeted vector? These
questions are still critical problems in tumor gene therapy.
Adenovirus is a medium-sized, icosahedral virus that contained a double-stranded
linear DNA genome. Adenovirus has many advantages include broad tissue tropism,
stability, safety, high lever expression and replication-defective(E1A and E1B deletion)
5
adenoviral vector can accommodate up to 7.5kb of foreign genes and can be amplified to
high titers in 293 cells. HIV-1 vpr is a 96-a.a. 14kDa protein associated with the HIV
virus particle. HIV-1 vpr has been shown to affect tumor cells in ways similar to that of
p53 and other tumor suppressor gene. Several laboratories have reported that vpr induces
apoptosis following induction of G2/M cell cycle arrest in some tumor lines. Pang et al.
reported that the mice of which the primary tumor were completely regressed by the vpr
were additionally protected from a secondary challenge of tumor cells. These results
suggest that the unique biological properties of vpr shown in some tumor lines suggest
that vpr may be a useful biological agent for anti-cancer therapy. Many cancers often
reexpress fetal or embryonic genes, and AFP gene expression is reactivated in HCC cells.
So we can use AFP promoter drive foreign gene expression to target AFP-producing
hepatoma cells to achieve hepatoma-specific gene therapy.
Targeting of tumor cells is crucial for gene therapy of malignant diseases. This can
be achieved by tumor-targeted gene transfer or tumor-specific gene expression. Base on
above consideration and theory, we use Ad5 pAdEasy system to generate recombinant
adenovirus by exploiting E.coli BJ5183 homologous recombination machinery. We
constructed a replication-defective adenoviral vector expressing vpr gene drived by AFP
promoter to achieve HCC-targeted gene therapy, named by rvAdAFP-vpr. We confirmed
that vpr gene and AFP promoter gene have been integrated into adenoviral genome by
PCR and Southern Blot methods.
In the present our study, non-AFP-producing hepatoma cell SMMC-7721 and
AFP-producing hepatoma cell BEL-7402 infected with rvAdAFP-vpr and the expression
of vpr protein in the infected cells was examined at 96h post infection. By flow
cytometer analysis, we found that vpr protein special expressed in the AFP-producing
hepatoma cell BEL-7402 and arrested BEL-7402 cell cycle at G2 phase, and we didn’t
6
find that expression of vpr protein and G2 cell cycle arrest in non-AFP-producing
hepatoma cell SMMC-7721. At the same time, we found that vpr protein could induce
BEL-7402 cell apoptosis by confocal microscopy. We made use of flow cytometer to
analysis activation of apoptosis-associated protein Bax, Caspase 3, Caspase 9, Caspase 8
and cytochrome c. Our results showed that high level Bax expression and activation of
Caspase 3, Caspase 9 and cytochro
细胞发生的癌。原发性肝癌是世界上恶性程度极高,且预后极差的恶性肿
瘤之一,每年全球约有 25 万人患病。在我国肝癌目前已处于恶性肿瘤死
亡率的第二位,每年至少有 10 万名新发现病人，11 万名肝癌病人死亡，
占全球肝癌死亡数的 45%。
尽管目前肝癌的治疗方法有许多种，但治疗效果都十分有限。大部
分肝癌病人对化疗和放疗均不敏感，因此寻找新的有效的治疗方式一直
是研究者追求的目标。随着分子生物学和免疫学理论及技术的不断发展
和对肿瘤发病机制认识的不断深入，利用基因工程手段对肝癌进行基因
治疗已取得了很大成功，日益受到研究者的重视。基因治疗及有可能成
为一种新的治疗方式应用于肿瘤的治疗之中。
腺病毒载体因其具有安全性好、外源基因表达效率高、滴度高、既可感
染分裂期细胞又可转染非分裂期细胞以及包装容量大等优点而成为当今
使用最多的病毒载体之一。
HIV vpr 基因是 I 型人类免疫缺陷病毒的非结构基因，编码一 14Kda
大小分子量的蛋白。研究表明，该蛋白是 HIV 重要的调控蛋白，Vpr 蛋
白不依赖于 HIV 其他蛋白的存在即可引起细胞的 G2 周期阻滞和诱导细
胞凋亡，单独表达的 Vpr 蛋白也可引起细胞的 G2 周期阻滞和诱导细胞凋
亡。文献报道 Vpr 蛋白可引起分裂细胞，包括肿瘤细胞发生凋亡。我们
1
的前期工作表明，HIV Vpr 对 Hela 细胞表现出明显的细胞周期阻滞作用。
由此可见 Vpr 很有可能作为一种新的治疗性基因而应用于以抑制肿瘤生
长为目的的基因治疗中。
目前在多数原发性肝细胞中有甲胎蛋白的表达，将外源治疗基因置
于 AFP 特异性启动子增强子下游，通过肝癌中 AFP 的特异性反式作用于
该启动子，以激活转录，可实现目的基因的选择性表达，从而可实现有
效的靶向性治疗，目前 AFP 启动子介导的肝癌特异性基因治疗已成为肝
癌特异性基因治疗领域的主要方面。
理想的基因治疗应具有目的基因高效性、靶向性转移和可控性表达
的特点。鉴于以上认识，我们应用非复制型腺病毒载体制备了甲胎蛋白
启动子控制下表达 HIV vpr 基因的重组腺病毒 rvAdAFP-vpr，实现其对肝
癌的特异性基因治疗作用，并对其生物学特征及其体外体内的治疗效果
进行研究，得到如下实验结果：
1．通过重组非复制型 5 型腺病毒 pAdEasy 系统，利用 E.coliBJ5183
的同源重组机制获得的甲胎蛋白启动子控制下表达 HIV vpr 基因重组非
复制型腺病毒 rvAdAFP-vpr，经 PCR 及 Southern blot 检测证实在重组腺
病毒基因组中整合有特异性的 HIV vpr 和 AFP 的上游调控序列。
2．重组腺病毒 rvAdAFP-Vpr 感染两种肝癌细胞：AFP 蛋白表达阳性
的 BEL-7402 细胞和 AFP 蛋白表达阴性的 SMMC-7721 细胞，实验结果表
明：Vpr 蛋白在 AFP 蛋白表达阳性的肝癌细胞 BEL-7402 细胞中特异性表
达，并且可引起 BEL-7402 细胞的细胞周期 G2 阻滞，G2 期细胞数目在病
毒感染后的 96 小时达到最大值，表现出一定的时间依赖性。对于 AFP
蛋白表达阴性的 SMMC-7721 细胞而言，却未能检测出 Vpr 蛋白的表达也
未见细胞周期阻滞作用。
3．通过荧光染色和和细胞线粒体膜电位改变等指标看到，重组腺病
2
毒 rvAdAFP-vpr 可诱导肝癌细胞凋亡。流式细胞术检测各个与细胞凋亡
有关的蛋白 Bax、Caspase 9、Caspase 8、Capase 3、细胞色素 C 表达情况。
结果显示，Bax 蛋白表达量明显升高，Caspase 9、Capase 3、细胞色素 C
等表达较对照都升高；而 caspase 8 蛋白表达未见明显变化。提示 Vpr 蛋
白可能通过线粒体途径引起细胞凋亡。
4．我们在裸鼠建立 AFP 蛋白表达阳性的人肝癌模型，并进行了 1
个月的分组治疗。免疫组化检测表明，rvAdAFP-vpr 可在肝癌组织中有效
表达 Vpr 蛋白。治疗效果表明，CPA 治疗组、rvAdAFP-vpr 治疗组和
rvAdAFP-vpr 联合 CPA 治疗组相对于空白对照组，都在不同程度上显著
抑制肿瘤生长，特别是 rvAdAFP-vpr 联合 CPA 组抑制肿瘤增长的作用更
为明显。CPA 治疗组、rvAdAFP-vpr 治疗组和 rvAdAFP-vpr 联合 CPA 治
疗组的抑瘤率分别达到了 41%、31%、66.7%，表明单独注射 rvAdAFP-vpr
组的抑瘤效果要好于 CPA 治疗组，说明单独注射 rvAdAFP-Vpr 即可一定
程度上起到抑制肿瘤生长的作用；另一方面，两者联合治疗组的抑瘤率
显著高于 rvAdAFP-vpr 组和 CPA 组的单独治疗，提示两者具有协同作用。
5．空白对照组和 rvAd-null 对照组的 Ki-67 指数明显高于 CPA 治疗
组、rvAdAFP-vpr 治疗组、rvAdAFP-vpr 联合 CPA 治疗组。其中对照组
和 rvAd-null 组的 Ki-67 指数对比于 rvAdAFP-vpr 联合 CPA 组具有统计学
意义，P<0.05。表明 Vpr 蛋白细胞周期 G2 期阻滞的作用有效地减缓了细
胞分裂增殖速度、抑制了肿瘤的快速增殖。CPA 治疗组、rvAdAFP-vpr
治疗组、rvAdAFP-vpr 联合 CPA 治疗组的凋亡指数明显高于对照组和
rvAd-null 组，其中 rvAdAFP-vpr 联合 CPA 组与对照组对比具有统计学意
义，P<0.05。体内实验?
Hepatomacellular carcimoma (HCC) is one of the most common malignancies
worldwide with an extremely poor prognosis. The major etiologic risk factors for HCC
development include toxin, hepatitis B virus (HBV) and hepatitis C virus(HCV) infection
as well as various inherited metabolic disorders. Surgical treatment is the most important
treatment of HCC. However, clinical observation have shown that tumor recurrence rates
are very high in patients with HCC who receive medical or surgical treatment. Thus new
treatment modalities must be pursued. With the expectation of increasing therapeutic
efficacy, gene therapy has been introduced as a new direction in treatment for HCC. Gene
therapy strategies against HCC include suppressor gene therapy, anti-sene gene therapy,
immunogene therapy, suicide gene therapy and combinanted-gene therapy. However, the
overall results of the studies are still disappointing. How to search for tumor-specific
agent of gene therapy? How to achieve appropriate spatial and temporal control of the
expression of the therapeutic gene? How to construct tumor tissue-targeted vector? These
questions are still critical problems in tumor gene therapy.
Adenovirus is a medium-sized, icosahedral virus that contained a double-stranded
linear DNA genome. Adenovirus has many advantages include broad tissue tropism,
stability, safety, high lever expression and replication-defective(E1A and E1B deletion)
5
adenoviral vector can accommodate up to 7.5kb of foreign genes and can be amplified to
high titers in 293 cells. HIV-1 vpr is a 96-a.a. 14kDa protein associated with the HIV
virus particle. HIV-1 vpr has been shown to affect tumor cells in ways similar to that of
p53 and other tumor suppressor gene. Several laboratories have reported that vpr induces
apoptosis following induction of G2/M cell cycle arrest in some tumor lines. Pang et al.
reported that the mice of which the primary tumor were completely regressed by the vpr
were additionally protected from a secondary challenge of tumor cells. These results
suggest that the unique biological properties of vpr shown in some tumor lines suggest
that vpr may be a useful biological agent for anti-cancer therapy. Many cancers often
reexpress fetal or embryonic genes, and AFP gene expression is reactivated in HCC cells.
So we can use AFP promoter drive foreign gene expression to target AFP-producing
hepatoma cells to achieve hepatoma-specific gene therapy.
Targeting of tumor cells is crucial for gene therapy of malignant diseases. This can
be achieved by tumor-targeted gene transfer or tumor-specific gene expression. Base on
above consideration and theory, we use Ad5 pAdEasy system to generate recombinant
adenovirus by exploiting E.coli BJ5183 homologous recombination machinery. We
constructed a replication-defective adenoviral vector expressing vpr gene drived by AFP
promoter to achieve HCC-targeted gene therapy, named by rvAdAFP-vpr. We confirmed
that vpr gene and AFP promoter gene have been integrated into adenoviral genome by
PCR and Southern Blot methods.
In the present our study, non-AFP-producing hepatoma cell SMMC-7721 and
AFP-producing hepatoma cell BEL-7402 infected with rvAdAFP-vpr and the expression
of vpr protein in the infected cells was examined at 96h post infection. By flow
cytometer analysis, we found that vpr protein special expressed in the AFP-producing
hepatoma cell BEL-7402 and arrested BEL-7402 cell cycle at G2 phase, and we didn’t
6
find that expression of vpr protein and G2 cell cycle arrest in non-AFP-producing
hepatoma cell SMMC-7721. At the same time, we found that vpr protein could induce
BEL-7402 cell apoptosis by confocal microscopy. We made use of flow cytometer to
analysis activation of apoptosis-associated protein Bax, Caspase 3, Caspase 9, Caspase 8
and cytochrome c. Our results showed that high level Bax expression and activation of
Caspase 3, Caspase 9 and cytochro
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