【摘要】：目的:草酸鈣結石是泌尿外科最常見的疾病之一,高草酸尿癥是其主要致病因素,目前尚無有效的防治方法。腸道吸收的食源性草酸是尿草酸的重要來源,減少食物中草酸含量可有效降低尿草酸排泄量。草酸脫羧酶(oxalate decarboxylase/ODC)和草酸氧化酶(oxalate oxidase/Ox O)是自然界中存在的兩種特異性草酸降解酶,在預防高草酸尿方面具有廣泛的應用前景。本研究將ODC和Ox O基因轉入乳酸菌(lactic acid bacteria/Lab)中,使其表達草酸降解酶,達到持續(xù)有效降解草酸的效果,通過口服途徑降解食物中的草酸,減少腸道草酸的吸收,降低尿草酸排泄量,從而預防草酸鈣結石的形成。方法:提取ODC及Ox O編碼基因,將基因片段與Lab表達質粒p MG36e相連接,為了提高表達效率,在編碼基因前插入p H誘導的強啟動子p170;將重組表達質粒通過電轉化轉入Lab菌株MG1363中。在含有100mmol/L草酸的MRS培養(yǎng)基中于37℃條件下靜置培養(yǎng)轉基因乳酸菌48h,利用分光光度計和高效液相色譜儀測量不同時間的培養(yǎng)基的吸光度和草酸濃度,檢測Lab的生長情況及降解草酸效率;用含5%草酸食物喂養(yǎng)SD大鼠,構建高草酸尿模型,同時轉基因乳酸菌灌胃治療1個月,分別在第0、7、14、21、28天收集24h尿液,測量尿液中草酸含量,并在第30天處死大鼠,取出腎臟,切片固定,利用硝酸銀染色,觀察腎臟中草酸鈣結晶形成情況。結果:轉ODC基因乳酸菌(ODC-Lab)和轉Ox O基因乳酸菌(Ox O-Lab)可在高草酸濃度培養(yǎng)基中生長,ODC-Lab可有效降解培養(yǎng)基中草酸,插入強啟動子p170(p170-ODC-Lab)可提高草酸降解效率;而Ox O-Lab和p170-Ox O-Lab并無有效降解草酸的功能;在動物實驗中,ODC-Lab和p170-ODC-Lab灌胃治療可明顯緩解高草酸尿并預防草酸鈣結石形成,其中p170-ODC-Lab效果更佳,相反Ox O-Lab和p170-Ox O-Lab無防治高草酸尿癥和草酸鈣結石的功效。結論:重組ODC乳酸菌可有效降解草酸,p170可以增強目的基因的生物學效應,ODC-Lab和p170-ODC-Lab都可通過口服途徑有效減少食源性草酸的吸收,并對預防草酸鈣結石具有顯著作用。
[Abstract]:Aim: calcium oxalate stone is one of the most common diseases in urology, hyperoxaluria is the main cause of disease, and there is no effective method to prevent and cure it. Food-derived oxalic acid absorbed by intestinal tract is an important source of urinary oxalic acid. Reducing oxalic acid content in food can effectively reduce urinary oxalic acid excretion. Oxalic acid decarboxylase (oxalate decarboxylase/ODC) and oxalate oxidase (oxalate oxidase/Ox O) are two kinds of specific oxalate degrading enzymes in nature. In this study, ODC and Ox O genes were transferred into Lactobacillus (lactic acid bacteria/Lab to express oxalic acid degrading enzyme, which could degrade oxalic acid continuously and effectively. The oxalic acid in food was degraded by oral administration, and the absorption of oxalic acid in intestinal tract was reduced. Reduce urinary oxalic acid excretion, thereby preventing the formation of calcium oxalate stones. Methods: ODC and Ox O genes were extracted and ligated with the Lab expression plasmid p MG36e. In order to improve the expression efficiency, the strong promoter p170 induced by pH was inserted before the coding gene. The recombinant expression plasmid was transformed into Lab strain MG1363 by electroporation. Transgenic lactic acid bacteria were statically cultured in MRS medium containing 100mmol/L oxalic acid at 37 鈩，

本文編號：2456768