【摘要】：一、目的：基因治療為骨性關(guān)節(jié)炎(Osteoarthritis, OA)的治療提供了可能性,目前該療法開(kāi)展臨床研究的瓶頸在于尋找一種安全有效的基因?qū)胪緩?本實(shí)驗(yàn)驗(yàn)證作為非病毒載體的電穿孔(Electroporation, EP)對(duì)骨性關(guān)節(jié)炎的治療效果,并與研究最為廣泛的腺相關(guān)病毒(Adeno-Associated Virus, AAV)載體作比較。二、方法：選用8周齡健康級(jí)雄性SD大鼠,在不同電穿孔參數(shù)下,使用電穿孔將熒光素酶(Luciferase)報(bào)告基因轉(zhuǎn)染到大鼠膝關(guān)節(jié)組織內(nèi),采用小動(dòng)物活體成像系統(tǒng)測(cè)定熒光強(qiáng)度,該強(qiáng)度反映在不同電穿孔參數(shù)下Luciferase基因表達(dá)水平,通過(guò)比較熒光強(qiáng)度以明確較佳的電穿孔參數(shù)。通過(guò)切斷右膝前交叉韌帶及切除內(nèi)側(cè)半月板前腳的方法建立骨性關(guān)節(jié)炎模型,在造模術(shù)后1周,按上述所選的較佳參數(shù)值進(jìn)行下一步大鼠體內(nèi)治療實(shí)驗(yàn)。選擇白細(xì)胞介素-1受體阻滯劑(Interleukin-1 receptor antagonist, IL-1Ra)作為目的基因,抑制由白細(xì)胞介素-1引起的炎癥反應(yīng)。根據(jù)不同的治療方式將實(shí)驗(yàn)分為五組：?jiǎn)渭児切躁P(guān)節(jié)炎未治療組(OA組,n=24)：骨性關(guān)節(jié)炎單純目的基因質(zhì)粒治療組(NP組,n=24);骨性關(guān)節(jié)炎電穿孔目的基因治療組(EP組,n=24);骨性關(guān)節(jié)炎腺相關(guān)病毒轉(zhuǎn)染目的基因治療組(AAV組,n=24);正常對(duì)照組(Normal, n=24)。治療后1周開(kāi)始評(píng)價(jià),每周取材1次,共計(jì)4次。取材前使用CatWalk進(jìn)行步態(tài)分析,了解由于炎癥引起的患肢疼痛情況;處死大鼠抽取右膝關(guān)節(jié)液進(jìn)行ELISA檢測(cè),分析關(guān)節(jié)液中IL-1Ra、IL-1β、 TNF-a蛋白含量變化;分離右膝關(guān)節(jié)股骨遠(yuǎn)端,進(jìn)行大體觀觀察,部分標(biāo)本立即進(jìn)行PCR檢測(cè),了解IL-1Ra、IL-1β、TNF-α、MMP-13、ADAMTS-4基因的表達(dá)情況,部分標(biāo)本進(jìn)行Micro-CT掃描以了解軟骨下骨的重建情況,掃描后的樣本進(jìn)行固定脫鈣、石蠟切片、病理染色,包括蘇木素-伊紅染色、番紅O染色、甲苯胺藍(lán)染色、IL-1Ra及Ⅱ型膠原的免疫組織化學(xué)染色。三、結(jié)果：在電穿孔參數(shù)脈寬為30ms,質(zhì)粒量為50μg時(shí),右側(cè)膝關(guān)節(jié)表達(dá)的熒光強(qiáng)度明顯高于其余各組(p0.05)。在EP組及AAV組,由炎癥反應(yīng)引起的疼痛水平明顯的低于OA組和NP組(p0.05),于軟骨缺損部位選取興趣區(qū),測(cè)得軟骨下骨骨礦化密度、骨小梁厚度升高明顯不及OA組和NP組(p0.05); PCR結(jié)果顯示：EP組及AAV組IL-1Ra基因表達(dá)上調(diào)明顯高于其余組(p0.05),前兩周顯示,AAV組的IL-1Ra表達(dá)量明顯的高于EP組,后兩周結(jié)果相反。在OA組和NP組，IL-1β、TNF-α表達(dá)明顯上調(diào)。EP組及AAV組IL-1β、TNF-α表達(dá)高于正常對(duì)照但無(wú)統(tǒng)計(jì)學(xué)差異(p0.05)。MMP-13和ADAMTS-4的變化趨勢(shì)類(lèi)似于IL-1p和TNF-α、ELISA結(jié)果顯示出與PCR相同的變化趨勢(shì);病理結(jié)果顯示：4周時(shí),EP組及AAV組未見(jiàn)明顯軟骨破壞及細(xì)胞外基質(zhì)丟失,表達(dá)IL-1Ra陽(yáng)性區(qū)域分布在軟骨細(xì)胞核周?chē)?表達(dá)Ⅱ型膠原陽(yáng)性區(qū)域主要分布在軟骨淺表層及中間層,兩組間無(wú)差異。OA組和NP組軟骨破壞嚴(yán)重,存留少許基質(zhì),局部區(qū)域呈現(xiàn)弱陽(yáng)性IL-1Ra表達(dá)、弱陽(yáng)性Ⅱ型膠原表達(dá)。四、結(jié)論：電穿孔即能獲得與腺相關(guān)病毒相當(dāng)?shù)闹委熜Ч帜茌^長(zhǎng)時(shí)間的表達(dá)目的基因,將安全性考慮在內(nèi),電穿孔可以替代病毒載體用于骨性關(guān)節(jié)炎基因治療的進(jìn)一步實(shí)驗(yàn)研究。
[Abstract]:1. Objective: To provide the possibility of gene therapy for the treatment of Osteoarthritis (OA). At present, the bottleneck of this therapy is to find a safe and effective gene introduction route, and this experiment verifies the electroporation as a non-viral vector (Electrotrop, The effect of EP on the treatment of osteoarthritic arthritis is compared with the most widely used adeno-associated virus (AAV) vector. 2. The method comprises the following steps of: selecting 8-week-old healthy male SD rats, using electroporation to transfect a luciferase reporter gene into a rat knee joint tissue under different electroporation parameters, and measuring the fluorescence intensity by using a small-animal living body imaging system; This intensity reflects the expression level of the Lucifasse gene under different electroporation parameters, and by comparing the fluorescence intensity to define better electroporation parameters. A model of osteogenic arthritis was established by cutting the anterior cruciate ligament of the right knee and the anterior foot of the medial meniscus. Interleukin-1 receptor blocker (IL-1Ra) was selected as the target gene to inhibit the inflammatory response induced by interleukin-1. The experiment was divided into five groups according to different treatment methods: the non-treated group (OA group, n = 24), the pure-purpose gene plasmid treatment group (NP group, n = 24), and the gene therapy group (EP group, n = 24). Gene therapy group (AAV group, n = 24) for transfection of bone-related virus-related virus; normal control group (Normal, n = 24). The evaluation was started 1 week after treatment, and once a week, four times were obtained. Cavalk was used for gait analysis to understand the pain of the patient with limb pain due to inflammation, and the right knee fluid was extracted by ELISA, and the content of IL-1Ra, IL-1 and TNF-a in the joint fluid was analyzed, and the distal end of the right knee joint was isolated, and the general observation was performed. The expression of IL-1Ra, IL-1, TNF-1, MMP-13 and ADAMTS-4 was detected by PCR. The microCT scan of some specimens was performed to understand the reconstruction of the subchondral bone. The immunohistochemical staining of O-staining, toluidine blue staining, IL-1Ra and type II collagen were studied. The results showed that when the pulse width of the electroporation parameters was 30ms and the amount of the plasmid was 50. m u.g, the intensity of the expression of the right knee joint was significantly higher than that of the other groups (p0.05). In EP group and AAV group, the level of pain caused by inflammatory reaction was lower than that in OA group and NP group (p0.05). The expression of IL-1Ra in the EP group and the AAV group was significantly higher than that of the other group (p0.05). The expression of IL-1Ra in the AAV group was significantly higher than that of the EP group in the first two weeks. In the OA and NP groups, the expression of IL-1 and TNF-1 was up-regulated. The expression of IL-1 and TNF-1 in EP group and AAV group was higher than that of normal control, but there was no statistical difference (p0.05). The change trend of MMP-13 and ADAMTS-4 was similar to that of IL-1p and TNF-1, and the results of ELISA showed the same trend as that of PCR. In EP group and AAV group, no significant cartilage destruction and extracellular matrix loss were found, and the expression of IL-1Ra positive region was distributed around the nucleus of the cartilage. The expression of type 鈪，

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