本文選題：溶瘤腺病毒 + 載體構(gòu)建��； 參考：《中國人民解放軍軍事醫(yī)學科學院》2008年博士論文

【摘要】： 溶瘤腺病毒載體(Oncolytic adenovectors),又稱條件復(fù)制型腺病毒載體(Conditionally replicating adenoviruses, CRADs),現(xiàn)已廣泛用于腫瘤基因治療研究中。CRADs名稱中條件復(fù)制的含義是指這種病毒載體能夠在腫瘤細胞中特異性復(fù)制,通過病毒擴增達到裂解、殺傷腫瘤細胞的目的,其在正常細胞中不能復(fù)制,對正常細胞無傷害或傷害小。傳統(tǒng)的腫瘤基因治療的策略是利用復(fù)制缺陷型的載體將治療基因轉(zhuǎn)導(dǎo)到腫瘤細胞,實踐證明這種方法由于基因轉(zhuǎn)移效率低,難以殺滅大多數(shù)腫瘤細胞。CRADs由于能夠在腫瘤細胞中復(fù)制、傳播,較好地克服了復(fù)制缺陷病毒載體的不足。一般認為,CRADs攜帶治療基因后,較不含治療基因的CRADs能夠更有效的殺傷腫瘤細胞。 CRADs的構(gòu)建通常采用穿梭質(zhì)粒和骨架質(zhì)粒在293細胞中同源重組、拯救獲得重組病毒的傳統(tǒng)方法。真核細胞內(nèi)重組發(fā)生的效率低,需要的時間長;為了排除污染、獲得均一的目的病毒,還需要進行噬斑純化操作。1998年,AdEasy系統(tǒng)的發(fā)明有效地克服了在上述傳統(tǒng)方法的不足,其通過穿梭質(zhì)粒和骨架質(zhì)粒在細菌中重組,方便快速地獲得重組病毒基因組DNA,轉(zhuǎn)染包裝細胞后能快速產(chǎn)生均一的重組病毒。AdEasy系統(tǒng)原本只能用于構(gòu)建復(fù)制缺陷型腺病毒載體,本研究對其進行了改造,建立了一種攜帶1-2個目的基因的CRAD構(gòu)建系統(tǒng),利用該系統(tǒng)構(gòu)建了攜帶治療基因的CRADs,觀察了體內(nèi)外的腫瘤殺傷效應(yīng)。 首先構(gòu)建一個攜帶腺病毒E1區(qū)的新質(zhì)粒pTE-TPE-GM,它具備三個特征:一、攜帶的5型腺病毒(Ad5)E1區(qū)序列,這將賦予溶瘤腺病毒選擇性復(fù)制的特性,二、其基因組較小,便于對E1區(qū)進行改造;三、E1區(qū)序列能夠方便的切下,并亞克隆到穿梭質(zhì)粒的合適部位。在這個新質(zhì)粒的E1區(qū)序列中,E1A的啟動子由腫瘤或組織特異型啟動子(TSP)替代,E1B19K序列由第1個治療基因替代,在治療基因和E1B55K之間添加內(nèi)部核糖體進入位點(IRES)序列。將改造的E1區(qū)切下,插入到穿梭質(zhì)粒pShuttle-CMV的腺苷酸加尾信號和腺病毒右臂序列之間,得到一個新的穿梭質(zhì)粒,其與AdEasy-1共轉(zhuǎn)化大腸桿菌BJ5183,同源重組生成了攜帶1個治療基因轉(zhuǎn)錄調(diào)控的溶瘤腺病毒載體。如果先在pShuttle-CMV的多克隆序列處插入第2個治療基因,再將改造的E1區(qū)引入這個穿梭質(zhì)粒,同理就能夠獲得攜帶2個治療基因的溶瘤腺病毒載體。選擇端粒酶啟動子(TERTp)控制E1A基因的表達,在成功構(gòu)建pTE-TPE-GM后,利用此系統(tǒng),設(shè)計并構(gòu)建了兩個溶瘤腺病毒,一個是攜帶粒細胞巨噬細胞集落刺激因子(GM-CSF)和細胞表面共刺激分子B7-1(CD80)的Ad-CD80-TPE-GM,另一個是攜帶GFP和GM-CSF的Ad-GFP-TPE-GM。本研究還構(gòu)建了復(fù)制缺陷腺病毒Ad-GFP,作為陰性對照。病毒構(gòu)建后,進行了功能檢測。 體外實驗中,檢測了Ad-CD80-TPE-GM和Ad-GFP-TPE-GM的在體外培養(yǎng)腫瘤細胞中的復(fù)制和選擇性腫瘤殺傷效應(yīng)。預(yù)實驗觀察到兩種溶瘤腺病毒能夠殺死端粒酶陽性的PC3M細胞,并證明病毒在其中復(fù)制。然后,選用8種端粒酶陽性腫瘤細胞系和2種端粒酶陰性細胞系,進一步評價載體的選擇性腫瘤殺傷效應(yīng)。將病毒感染不同的細胞系,感染后7d,結(jié)晶紫染色法檢測細胞存活百分數(shù),實驗結(jié)果表明Ad-CD80-TPE-GM和Ad-GFP-TPE-GM均能夠高效特異地殺傷端粒酶陽性腫瘤細胞,以Ad-CD80-TPE-GM的溶瘤效果更為顯著。在10MOI感染條件下,Ad-CD80-TPE-GM能夠殺死幾乎所有端粒酶陽性腫瘤細胞,在1MOI感染條件下,Ad-CD80-TPE-GM能夠殺死80%的端粒酶陽性腫瘤細胞,甚至在0.1 MOI感染條件下,Ad-CD80-TPE-GM的溶瘤效果也非常明顯。同時兩病毒對端粒酶陰性的正常細胞沒有明顯的殺傷作用。Ad-CD80-TPE-GM能夠選擇性地在端粒酶陽性腫瘤細胞中復(fù)制,每個細胞約可產(chǎn)生375感染單位(Infectious units,IU)的子代病毒。 體外實驗中,以復(fù)制缺陷病毒Ad-p53/GM-CSF/B7-1做對照,我們還檢測了Ad-CD80-TPE-GM介導(dǎo)目的基因CD80和GM-CSF的表達。在5MOI感染條件下,Ad-CD80-TPE-GM感染端粒酶陽性人喉癌細胞Hep2,培養(yǎng)48h,培養(yǎng)上清中GM-CSF的表達量達到100ng/ml。與對照相比,GM-CSF的表達增加了9000多倍。同時細胞表面CD80的表達陽性率接近100%,相對平均熒光強度與對照相比,增加了146倍。 在體內(nèi)裸鼠荷瘤實驗中,我們檢測了溶瘤腺病毒Ad-CD80-TPE-GM的功能。選擇BALB/C裸鼠,皮下接種端粒酶陽性的人喉癌細胞Hep2,建立荷瘤裸鼠模型,進行了動物實驗。以Ad-GFP做對照,瘤內(nèi)注射溶瘤腺病毒Ad-CD80-TPE-GM 1×10~9 IU,可明顯抑制腫瘤生長,給藥后35天,腫瘤抑制率達到了78%,血清轉(zhuǎn)氨酶沒有升高。瘤內(nèi)注射溶瘤腺病毒Ad-CD80-TPE-GM 1×10~9 IU,給藥后4天,腫瘤組織中可檢測到CD80和GM-CSF的表達,以及病毒的復(fù)制。 總之,本研究建立了一種攜帶1-2個外源基因溶瘤腺病毒載體生成系統(tǒng),在此系統(tǒng)中,組織特異性啟動子和治療基因均可根據(jù)需要進行相應(yīng)替換;利用該系統(tǒng)成功構(gòu)建了攜帶CD80和GM-CSF的溶瘤腺病毒載體Ad-CD80-TPE-GM,體內(nèi)外實驗表明其有較好的腫瘤殺傷作用。為利用CRADs進行腫瘤基因治療研究建立了平臺。
[Abstract]:Oncolytic adenovirus vector (Oncolytic adenovectors), also called conditionally replicative adenovirus (Conditionally replicating, adenoviruses, CRADs), which has been widely used in the study of meaning of.CRADs conditional replication in cancer gene therapy in the name refers to the virus vector specific replication in tumor cells by virus amplification to lysis, killing tumor cells the purpose, it cannot replicate in normal cells to normal cells, no injury or damage. The traditional gene therapy strategy is the use of vector replication defective gene therapy will be transferred to tumor cells, it is proved that this method is due to low gene transfer efficiency, it is difficult to kill most tumor cells due to.CRADs replication in tumor cell spreading, overcomes the defects of replication defective viral vectors. It is generally considered that CRADs gene carrying therapy, is free CRADs, a therapeutic gene, can kill tumor cells more effectively.
The construction of CRADs usually adopts the shuttle plasmid and plasmid homologous recombination in 293 cells, save the traditional method to obtain the recombinant virus. The recombination in eukaryotic cells is low efficiency, the need for a long time; in order to eliminate the pollution, to obtain a uniform objective virus, but also the need for plaque purification operation in.1998, the invention of AdEasy system to effectively overcome the shortcomings in the traditional method, the shuttle plasmid and the recombinant plasmid in bacteria, fast and convenient to obtain recombinant virus genomic DNA, recombinant virus.AdEasy transfected into the packaging cell can quickly produce uniform originally can only be used to construct replication defective adenovirus vector, this study was carried out on the transformation. A carry 1-2 CRAD gene to construct the system, construct carry the therapeutic gene CRADs by using this system, to observe the in vivo tumor killing effect.
First, build a new area of adenovirus carrying E1 plasmid pTE-TPE-GM, it has three characteristics: first, type 5 adenovirus carrying the E1 sequence (Ad5), which will give the characteristics of selective replication oncolytic adenovirus two, its genome is small, easy to modify E1; three, E1 sequence easy to cut, and subcloned into the shuttle plasmid. The appropriate parts of the E1 sequences in this new plasmid, E1A promoter by tumor or tissue specific promoter (TSP), E1B19K sequence from first for gene replacement, add in the treatment of internal ribosome entry site between gene and E1B55K (IRES) sequence. E1 will transform the region, into the polyadenylation signals and Adenovirus Shuttle Plasmid pShuttle-CMV sequence of the right arm, get a new shuttle plasmid and AdEasy-1 were transformed into Escherichia coli BJ5183, homologous recombination was generated carrying 1 treatment base Because of the oncolytic adenovirus vector of transcriptional regulation. If the first into the second gene therapy in the cloning sequence at pShuttle-CMV, then the E1 transformation of the region into the shuttle plasmid, oncolytic adenovirus vector can be obtained similarly carrying 2 gene therapy. Telomerase promoter (TERTp) to control the expression of E1A gene in. The successful construction of the pTE-TPE-GM, using this system, the design and construction of two oncolytic adenovirus carrying a granulocyte macrophage colony stimulating factor (GM-CSF) and cell surface costimulatory molecule B7-1 (CD80) Ad-CD80-TPE-GM, the other one is carrying GFP and GM-CSF Ad-GFP-TPE-GM. this study also constructed a replication defective adenovirus Ad-GFP virus, virus as negative control. After construction, the function of detection.
In vitro experiments, the detection of Ad-CD80-TPE-GM and Ad-GFP-TPE-GM in cultured tumor cells in tumor selective replication and cytotoxicity in vitro. The pre experiment observed two kinds of oncolytic adenovirus can kill telomerase positive PC3M cells, and prove that the virus replicates in them. Then, selected 8 kinds of telomerase positive tumor cell lines and 2 telomerase negative cell line, tumor selective killing effect. Further evaluation vector to cell lines of different virus infection after infection, 7d, crystal violet staining method to detect cell survival percentage, the experimental results show that Ad-CD80-TPE-GM and Ad-GFP-TPE-GM can effectively and specifically kill telomerase positive tumor cells by oncolytic Ad-CD80-TPE-GM effect is more significant in the 10MOI infection condition. Ad-CD80-TPE-GM, can kill almost all telomerase positive tumor cells in the 1MOI infection condition, Ad-CD80-TPE-GM can kill Telomerase positive tumor cells die 80%, even at 0.1 MOI under the condition of Ad-CD80-TPE-GM infection, the oncolytic effect is very obvious. At the same time two virus of normal cells telomerase negative no obvious cytotoxicity of.Ad-CD80-TPE-GM can selectively replicate in telomerase positive tumor cells, each cell can produce about 375 infectious units (Infectious units, IU) the progeny virus.
In vitro, the replication defective virus Ad-p53/GM-CSF/B7-1 control, we also examined the expression of Ad-CD80-TPE-GM gene mediated by CD80 and GM-CSF. In the 5MOI infection condition, cultured 48h telomerase positive human laryngeal carcinoma cell Hep2, Ad-CD80-TPE-GM infection and the expression level of culture supernatant reached GM-CSF 100ng/ml. compared with the control group, the expression of GM-CSF increased by 9000 at the same time times. The positive expression rate of cell surface CD80 close to 100%, the relative mean fluorescence intensity compared with the control, increased by 146 times.
In vivo nude mice, we tested the oncolytic adenovirus Ad-CD80-TPE-GM function. BALB/C nude mice subcutaneous inoculation of telomerase positive Hep2 human laryngeal carcinoma cells, the establishment of nude mice model, animal experiments were carried out. In order to control Ad-GFP, intratumoral injection of oncolytic adenovirus Ad-CD80-TPE-GM 1 * 10~9 IU, can inhibit tumor growth, 35 days after the administration, the tumor inhibition rate reached 78%, serum transaminase increased. No intratumoral injection of oncolytic adenovirus Ad-CD80-TPE-GM 1 * 10~9 IU, 4 days after administration, detected CD80 and GM-CSF expression in tumor tissues, and the replication of the virus.
In conclusion, this study established a 1-2 carrying exogenous gene oncolytic adenovirus vector generation system, in this system, tissue specific promoter and gene therapy can be carried out according to the needs of the corresponding replacement; successfully constructed oncolytic adenovirus vector carrying Ad-CD80-TPE-GM CD80 and GM-CSF by using this system, in vitro and in vivo experiments show that it has better the tumor killing effect. For the use of CRADs to establish a platform for the research of tumor gene therapy.

【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R346;R73-3

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