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人乳頭瘤病毒16型(HPV16)E7 DNA疫苗及微針陣列透皮給藥的研究

發(fā)布時間:2018-08-04 21:07
【摘要】:人乳頭瘤病毒(human papillomavirus,HPV)是乳多空病毒的一種,生殖道HPV感染是當(dāng)今流行最廣的性傳播疾病之一。HPV在臨床上主要引起尖銳濕疣和宮頸癌等疾病,它的型別繁多,現(xiàn)已發(fā)現(xiàn)200多個,根據(jù)其致病力的大小分為低危型和高危型。前者包括HPV6,11,30,9等,主要導(dǎo)致生殖道乳頭狀瘤,尖銳濕疣,外生性濕疣類病變,低度子宮頸上皮內(nèi)瘤變等。后者包括HPV16,18,31,33,35等,主要導(dǎo)致高度子宮頸上皮內(nèi)瘤變和子宮頸癌的發(fā)生。根據(jù)研究發(fā)現(xiàn)子宮頸癌的發(fā)生與HPV的感染密切相關(guān),90%以上的宮頸癌組織中可以檢測到HPV,其中HPV16的檢測率超過50%。所以可以說,HPV特別是HPV16是引起宮頸癌的主要因素。 近來有一種新的給藥方式,它利用在顯微鏡下才看得見的針組成的微針陣列(microneedle array,簡稱微針,)在皮膚上產(chǎn)生許多微米級的通道。這些通道可以讓大分子物質(zhì)通過,甚至一些超分子化合物和微粒也能通過。微針僅穿透對藥物擴(kuò)散起屏障作用的皮膚角質(zhì)層(15~25μm),并打通上百個給藥通道,藥物可經(jīng)通道迅速擴(kuò)散到毛細(xì)血管進(jìn)入體循環(huán)。而且微針透皮給藥方便,無明顯創(chuàng)傷,病人痛苦小,可減少副作用,同時設(shè)備操作簡單,價格便宜。作為抵御外界病源的第一道屏障,皮膚系統(tǒng)的免疫功能十分發(fā)達(dá),能否通過透皮免疫引起有效的免疫反應(yīng),以建立一種新型的免疫途徑,正是我們的研究目的。 本文構(gòu)建了HPV16E7—pcDNA3.1(-)重組質(zhì)粒,并以之作為DNA疫苗。在體外透皮的條件下,我們觀察了30個小時,試驗結(jié)果發(fā)現(xiàn)DNA疫苗可以透過皮膚,而且透過量隨著時間的延長而逐步增加,透過量也比較大,在第30h時可以達(dá)到0.74mg/cm~2。再結(jié)合微針給藥的方式將該DNA疫苗免疫BALB/c小鼠,每兩周免疫一次,共給藥三次,每次免疫劑量為200μg/只,陰性對照組用等體積生理鹽水代替。最后一次免疫后兩周采血,分別分離血清和淋巴細(xì)胞,用間接免疫熒光試驗檢測小鼠的體液免疫功能,用淋巴細(xì)胞轉(zhuǎn)化實驗來檢測小鼠的細(xì)胞免疫功能。實驗結(jié)果表明,微針免疫DNA疫苗可以誘導(dǎo)小鼠產(chǎn)生體液免疫和細(xì)胞免疫。 本文通過HPV16 E7的DNA疫苗的研制為HPV的治療尋找一種有效的方法,同
[Abstract]:Human papillomavirus (human) is a kind of lactobacillus virus. Genital tract HPV infection is one of the most prevalent sexually transmitted diseases. HPVs mainly cause condyloma acuminatum and cervical cancer in clinic. According to its pathogenicity, it can be divided into low risk type and high risk type. The former included HPV6, 1130T9, which mainly resulted in genital papilloma, condyloma acuminatum, ectogenic condyloma, low grade cervical intraepithelial neoplasia and so on. The latter, including HPV16, 18, 31, 33, 35, mainly lead to high cervical intraepithelial neoplasia and cervical cancer. According to the study, HPV can be detected in more than 90% of cervical cancer tissues, and the detection rate of HPV16 is more than 50%. So it can be said that HPV, especially HPV16, is the main factor causing cervical cancer. Recently, a new method of drug delivery has been developed, which uses microneedle array (microneedle array, which is visible under microscope, to produce many micron channels in the skin. These channels allow macromolecules to pass through, even supramolecular compounds and particles. The microneedle penetrates only the cuticle (1525 渭 m), which acts as a barrier to drug diffusion, and opens hundreds of drug delivery channels, through which the drug can rapidly spread to the capillary and enter the systemic circulation. And the microneedle transdermal drug is convenient, no obvious trauma, the patient pain is small, can reduce the side effect, at the same time the equipment is simple to operate, the price is cheap. As the first barrier to resist external diseases, the immune function of skin system is very developed. Whether transdermal immunity can induce an effective immune response to establish a new immune pathway is our research goal. HPV16E7-pcDNA3.1 (-) recombinant plasmid was constructed and used as DNA vaccine. Under the condition of in vitro transdermal penetration, we observed for 30 hours, the results showed that the DNA vaccine could penetrate the skin, and the transmissivity increased gradually with time, and the transmissivity reached 0.74 mg / cm ~ (-2) at 30 h. BALB/c mice were immunized with the DNA vaccine once every two weeks with a dose of 200 渭 g / mouse. The negative control group was replaced by the same volume of normal saline. Blood samples were collected two weeks after the last immunization. Serum and lymphocytes were separated respectively. The humoral immune function of mice was detected by indirect immunofluorescence test and the cellular immune function of mice was detected by lymphocyte transformation test. The results showed that microneedle immunized DNA vaccine could induce humoral and cellular immunity in mice. Through the development of DNA vaccine of HPV16 E7, an effective method for the treatment of HPV was found.
【學(xué)位授予單位】:揚州大學(xué)
【學(xué)位級別】:碩士
【學(xué)位授予年份】:2005
【分類號】:R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 解洪業(yè);基因疫苗的研究近況[J];青海畜牧獸醫(yī)雜志;2004年02期

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本文編號:2165147

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