高胰島素處理相關(guān)核酸適體的篩選及基于核酸適體技術(shù)的胰島素檢測(cè)
發(fā)布時(shí)間：2018-08-11 11:34
【摘要】：第一部分建立針對(duì)肝細(xì)胞的指數(shù)級(jí)富集的配體系統(tǒng)進(jìn)化技術(shù) 目的：建立針對(duì)肝細(xì)胞的指數(shù)級(jí)富集的配體系統(tǒng)進(jìn)化技術(shù)(Systematic Evolution of Ligands by Exponential Enrichment, SELEX),并獲得能識(shí)別肝細(xì)胞表面膜蛋白的核酸適體(Aptamer)。 方法：設(shè)計(jì)DNA文庫(kù),兩端各一段長(zhǎng)20bp的引物序列,引物1用異硫氰酸熒光素(fluorescein isothiocyanate, FITC)標(biāo)記,引物2用生物素biotin標(biāo)記,中間20bp為隨機(jī)序列。待培養(yǎng)皿(100mm)中人肝細(xì)胞癌細(xì)胞株HepG2生長(zhǎng)豐度達(dá)到95%左右時(shí),棄去細(xì)胞培養(yǎng)基,用4℃洗滌緩沖液洗滌。取20nmole DNA文庫(kù),溶于1ml結(jié)合緩沖液中,與細(xì)胞在4℃孵育1h后棄去上清液,洗滌緩沖液洗滌兩次后,用細(xì)胞刮子收集細(xì)胞,用1ml蒸餾水洗滌,并將細(xì)胞懸液收集在1.5ml離心管中,在95℃加熱15min后離心,取上清液,此即首輪篩選的母液,其內(nèi)包含與HepG2結(jié)合的序列。經(jīng)過(guò)聚合酶鏈?zhǔn)椒磻?yīng)(PCR)擴(kuò)增所得序列,擴(kuò)增后的產(chǎn)物為雙鏈DNA,包含目標(biāo)鏈及其互補(bǔ)鏈。將PCR產(chǎn)物與鏈親素(Streptavidin)覆蓋的瓊脂糖珠孵育后濾過(guò)上清液,用2%NaOH打開(kāi)PCR雙鏈結(jié)構(gòu),目標(biāo)鏈則溶解于NaOH溶液中,將該溶液通過(guò)NAP-5脫鹽柱脫去NaOH,測(cè)定單鏈DNA (ssDNA)濃度以確定該輪篩選終產(chǎn)物總量,并將其甩干。從上一輪篩選終產(chǎn)物中取100pmole,并將其溶于500ul含有10%胎牛血清的結(jié)合緩沖液中,將其與培養(yǎng)皿(60mm)中生長(zhǎng)豐度達(dá)到95%左右的HepG2細(xì)胞孵育,按照上述方法完成每一輪篩選。每3-5輪篩選后需要檢查每輪產(chǎn)物與細(xì)胞的結(jié)合情況。待細(xì)胞生長(zhǎng)豐度達(dá)95%左右時(shí),用非酶消化液消化細(xì)胞,將貼壁生長(zhǎng)的細(xì)胞配成單個(gè)細(xì)胞懸液,用洗滌緩沖液洗滌后將細(xì)胞溶于結(jié)合緩沖液中,每5×105個(gè)細(xì)胞與25pmole的篩選產(chǎn)物于4℃孵育30min,篩選產(chǎn)物的濃度為250nM。同時(shí)將細(xì)胞與同等濃度的DNA文庫(kù)孵育,作為陰性對(duì)照。孵育結(jié)束后洗滌細(xì)胞,用流式細(xì)胞儀分析篩選產(chǎn)物與細(xì)胞的結(jié)合程度。隨著篩選輪數(shù)的增加,產(chǎn)物與細(xì)胞間的結(jié)合程度越來(lái)越高,直至達(dá)到平臺(tái)期。選取結(jié)合程度最高的三輪產(chǎn)物進(jìn)行測(cè)序,所得序列即針對(duì)靶細(xì)胞的核酸適體。用DNA合成儀合成所得序列并用流式細(xì)胞儀檢測(cè)各條序列與靶細(xì)胞的結(jié)合程度,從而確定本篩選最終所得的核酸適體。分別用胰酶以及蛋白酶K處理細(xì)胞,處理時(shí)間從5min-30min不等,觀察酶處理前后各核酸適體與細(xì)胞結(jié)合的情況。同時(shí),將各核酸適體與不同種類的細(xì)胞孵育(包括LH86、Huh7、WT、IRS/KO、M7617、Ramos、CEM、H23、H69、A549、HBE、H661、TOV-21G、CAOV3等細(xì)胞),觀察各序列的結(jié)合情況。 結(jié)果：本文通過(guò)應(yīng)用SELEX對(duì)HepG2細(xì)胞展開(kāi)篩選,經(jīng)過(guò)測(cè)序分析發(fā)現(xiàn)了4條針對(duì)HepG2細(xì)胞的核酸適體：IR01、IR03、IR04以及IR06。其中IR01、IR04與靶細(xì)胞的親和性最佳,其解離常數(shù)分別為11.287±3.786nM和88.849±22.339nM; IR03雖然與HepG2細(xì)胞結(jié)合程度較低,解離常數(shù)較高(129.513±47.924nM),但其特異性最好,能特異性與HepG2細(xì)胞結(jié)合,而與其它種類細(xì)胞幾乎無(wú)交叉反應(yīng)；IR01、IR04、IR06對(duì)大部分肝臟來(lái)源的細(xì)胞株均有較好結(jié)合；IR01是本實(shí)驗(yàn)所得的核酸適體當(dāng)中與靶細(xì)胞結(jié)合程度最強(qiáng)的一條序列,其僅與肝臟來(lái)源的細(xì)胞有結(jié)合,而與其他組織來(lái)源的細(xì)胞無(wú)交叉反應(yīng),并且該條核酸適體與細(xì)胞的結(jié)合不容易被蛋白酶的作用所破壞。相反,IR03, IR04, IR06三條核酸適體與靶細(xì)胞的結(jié)合均容易被酶的消化而破環(huán),胰酶或者蛋白酶K處理HepG2細(xì)胞5min后上述三條核酸適體與靶細(xì)胞的結(jié)合即被完全破壞。 結(jié)論：通過(guò)SELEX,可以獲得與靶細(xì)胞高親和性以及高特異性結(jié)合的核酸適體,這些核酸適體通過(guò)識(shí)別靶細(xì)胞膜上所表達(dá)的某種生物分子從而達(dá)到識(shí)別表達(dá)相同生物分子的不同細(xì)胞株。 第二部分篩選針對(duì)肝細(xì)胞表面與高胰島素處理相關(guān)膜蛋白的核酸適體 目的：在第一部分實(shí)驗(yàn)的基礎(chǔ)上篩選出針對(duì)肝細(xì)胞表面與高胰島素處理相關(guān)膜蛋白的核酸適體(Aptamer)。 方法：待HepG2細(xì)胞生長(zhǎng)豐度達(dá)70%左右后血清饑餓12h,然后分成四組,第一組繼續(xù)予以不含胰島素的培養(yǎng)基培養(yǎng),第二組予以加入生理劑量的胰島素(0.1nM)的培養(yǎng)基培養(yǎng),第三組予以加入高胰島素(100nM)的培養(yǎng)基培養(yǎng),第四組予以加入超高胰島素(500nM)的培養(yǎng)基培養(yǎng)。同時(shí),予以高胰島素(100nM)處理胎鼠肝細(xì)胞野生型(Wild type, WT)與胰島素受體底物2基因(Insulin receptor substrate 2, IRS2)敲除后的胎鼠肝細(xì)胞(IRS2/KO),并設(shè)立正常培養(yǎng)組(不加入胰島素處理)作為對(duì)照。上述各組細(xì)胞的培養(yǎng)基中均不含血清。24h后用非酶消化液消化細(xì)胞,將貼壁生長(zhǎng)的細(xì)胞配成單個(gè)細(xì)胞懸液,用洗滌緩沖液洗滌后將細(xì)胞溶于結(jié)合緩沖液中,每5×105個(gè)細(xì)胞與濃度為250nM的核酸適體25pmole于4℃孵育30min。孵育結(jié)束后洗滌細(xì)胞,用流式細(xì)胞儀分析各核酸適體與上述各細(xì)胞的結(jié)合程度在高胰島素處理前后有無(wú)變化。 結(jié)果：IR04與HepG2細(xì)胞的結(jié)合明顯受到100nM胰島素的抑制；進(jìn)一步加大胰島素的劑量(500nM)則抑制作用更加明顯,經(jīng)過(guò)500nM胰島素處理后IR04與HepG2的結(jié)合幾乎被完全消減；但I(xiàn)R04與HepG2細(xì)胞的結(jié)合不受生理劑量胰島素的影響。同樣,高胰島素處理也能減弱IR04與WT細(xì)胞的結(jié)合；但I(xiàn)R04與IRS2/KO細(xì)胞的結(jié)合程度不受高胰島素處理的影響。而IR01、IR03、IR06與細(xì)胞的結(jié)合均不受高胰島素處理的影響。 結(jié)論：IR04與靶細(xì)胞的結(jié)合被高胰島素處理所減弱,且其減弱的程度與高胰島素的濃度呈正相關(guān)；但生理濃度范圍內(nèi)的胰島素不影響IR04與靶細(xì)胞的結(jié)合；IR04可能的靶標(biāo)為肝細(xì)胞表面某種與胰島素作用相關(guān)的膜蛋白,此種膜蛋白在高胰島素處理過(guò)程中被下調(diào),但具體機(jī)制有待進(jìn)一步研究；IR04所針對(duì)的靶物質(zhì)在人、鼠肝細(xì)胞的表達(dá)具有高度同源性,且其受高胰島素影響而發(fā)生下調(diào)的機(jī)理可能與人類相似。 第三部分基于核酸適體修飾石墨烯的胰島素檢測(cè) 目的：應(yīng)用核酸適體修飾后的氧化石墨烯(GO)來(lái)進(jìn)行胰島素的檢測(cè),并通過(guò)加入催化劑(DNA酶)來(lái)實(shí)現(xiàn)其檢測(cè)信號(hào)的放大。 方法：將天然石墨粉和氯化鈉結(jié)晶研磨從而減小石墨顆粒的體積。去掉氯化鈉后,將研磨過(guò)的石墨粉加入到濃硫酸中,強(qiáng)力攪拌下加入高錳酸鉀,并用體積分?jǐn)?shù)3%的雙氧水還原剩余的高錳酸鉀和二氧化錳,使其變?yōu)闊o(wú)色可溶的硫酸錳。在雙氧水的處理下,懸浮液變成亮黃色。最后,過(guò)濾、洗滌3次,然后超聲處理4h,離心后取上層溶液用于下一步實(shí)驗(yàn)。 異硫氰酸熒光素(FITC)標(biāo)記的胰島素核酸適體(insulin binding aptamer, IBA)稀釋成100nM的胰島素緩沖液。將IBA與GO按照摩爾濃度1：1于室溫條件下混勻,避光孵育30min。此即本實(shí)驗(yàn)的工作溶液。由于GO能淬滅吸附在其表面的IBA所攜帶的熒光,此時(shí)溶液中僅存在一個(gè)較弱的熒光信號(hào)。當(dāng)溶液中存在胰島素時(shí),與GO結(jié)合的IBA則從GO表面解離,與胰島素結(jié)合,FITC標(biāo)記的IBA游離到溶液中,GO對(duì)FITC的淬滅作用大大減弱,從而使得溶液中的熒光信號(hào)增加；進(jìn)一步在工作溶液中加入DNA酶,加入不同濃度的胰島素(胰島素的加入量從5nM到50μM)后孵育2h。此時(shí)與胰島素結(jié)合的IBA將被DNA酶消化成片段,從而失去與胰島素結(jié)合的能力,胰島素重新游離于工作液中；而與GO結(jié)合的IBA由于被GO保護(hù),不被DNA酶消化,但將會(huì)從GO表面解離,并與游離的胰島素結(jié)合,從而被DNA酶消化成片段。理論上該反應(yīng)可以無(wú)限循環(huán)至IBA耗盡。終止反應(yīng)后檢測(cè)溶液中熒光濃度。同時(shí)設(shè)立陰性對(duì)照組(生物素、鏈霉親和素、牛血清蛋白)以明確上述胰島素檢測(cè)體系的特異性。 結(jié)果：本實(shí)驗(yàn)通過(guò)將胰島素核酸適體修飾在氧化石墨烯單層表面,成功構(gòu)建了胰島素的生物感應(yīng)器,實(shí)現(xiàn)了胰島素的便捷檢測(cè),檢測(cè)下限為500nM；通過(guò)進(jìn)一步加入DNA酶來(lái)實(shí)現(xiàn)信號(hào)放大后,胰島素的檢測(cè)下限降低到5nM。 結(jié)論：胰島素核酸適體修飾后的氧化石墨烯可以作為胰島素檢測(cè)的良好工具,通過(guò)加入DNA酶實(shí)現(xiàn)檢測(cè)信號(hào)的放大,將檢測(cè)下限降低了100倍。
[Abstract]:The first part is to establish a system of phylogeny for exponential enrichment of hepatocytes.
OBJECTIVE: To establish a ligand system evolution technique (SELEX) for exponential enrichment of hepatocytes, and to obtain an aptamer (Aptamer) for recognizing hepatocyte surface membrane proteins.
METHODS: DNA libraries were designed with 20 BP primer sequences at each end. Primer 1 was labeled with fluorescein isothiocyanate (FITC), primer 2 was labeled with biotin, and the intermediate 20 BP was a random sequence. The cells were washed twice with the washing buffer. The cells were collected with a cell scraper and washed with 1 ml distilled water. The cell suspension was collected in a 1.5 ml centrifugal tube. The supernatant was centrifuged at 95 C for 15 minutes. The mother liquor of the round-screening contains the sequence bound to HepG2. The amplified product is double-stranded DNA containing target and complementary chains. The PCR product is incubated with streptavidin-coated agarose beads and the supernatant is filtered. The double-stranded structure of the PCR is opened with 2% NaOH, and the target chain is dissolved. NaOH was removed from NaOH solution by NAP-5 desalination column. Single-stranded DNA (ssDNA) concentration was determined to determine the total amount of the final screening product and then dried. 100 pmole was extracted from the final screening product and dissolved in a combination buffer containing 10% fetal bovine serum at 500 ul. The growth abundance of the final product in the culture dish (60mm) was 95%. After every 3-5 rounds of screening, we need to check the binding of the products to the cells. When the cell growth abundance reaches about 95%, the cells are digested with non-enzymatic digestive juice. The adherent cells are prepared into a single cell suspension and washed with a washing buffer to dissolve the cells into a combination. In the buffer solution, the screening products of 25 pmole and 5 *105 cells were incubated at 4 C for 30 min and the concentration of screening products was 250 nM. At the same time, the cells were incubated with DNA libraries of the same concentration as negative control. After incubation, the cells were washed and the binding degree between screening products and cells was analyzed by flow cytometry. Three rounds of products with the highest binding degree were selected for sequencing, and the sequence was aptamer for target cells. The DNA synthesized by DNA synthesizer and the binding degree between each sequence and target cells was detected by flow cytometry to determine the final nucleic acid. Aptamers were incubated with trypsin and protease K for 5 min to 30 min, respectively. The aptamers were observed before and after treatment. At the same time, aptamers were incubated with different kinds of cells (including LH86, Huh7, WT, IRS/KO, M7617, Ramos, CEM, H23, H69, A549, HBE, H661, TOV-21G, CAOV3, etc.) and observed. The combination of sequences.
RESULTS: Four aptamers, IR01, IR03, IR04 and IR06, were identified by SELEX. Among them, IR01, IR04 had the best affinity with target cells, and their dissociation constants were 11.287 (+ 3.786nM) and 88.849 (+ 22.339nM), respectively. The dissociation constant was high (129.513 47.924nM), but its specificity was the best, and it could bind to HepG2 cells with specificity and hardly cross-react with other kinds of cells; IR01, IR04, IR06 had good binding to most of the hepatic cell lines; IR01 was the most binding sequence between the aptamer and the target cells. In contrast, the binding of three aptamers IR03, IR04 and IR06 to target cells is easily broken by enzymatic digestion, and the binding of aptamers to target cells is treated by trypsin or protease K. The binding of these three aptamers to target cells was completely destroyed after HepG2 cells 5min.
Conclusion: High affinity and specific binding aptamers with target cells can be obtained by SELEX. These aptamers can identify different cell lines expressing the same biological molecules by recognizing some biological molecules expressed on the target cell membrane.
The second part screened aptamers targeting hepatocyte surface associated with high insulin processing membrane proteins.
AIM: To screen aptamers (Aptamers) targeting the membrane proteins associated with hyperinsulinemia on the surface of hepatocytes.
Methods: After the growth of HepG2 cells was about 70%, the serum was starved for 12 hours, and then divided into four groups. The first group continued to be cultured in insulin-free medium, the second group was cultured in a physiological dose of insulin (0.1nM), the third group was cultured in a high insulin (100nM) medium, and the fourth group was cultured in a super-high insulin (100nM) medium. At the same time, the wild type (WT) and insulin receptor substrate 2 (IRS2) knockout fetal rat hepatocytes (IRS2 / KO) were treated with high insulin (100nM) and the normal culture group (without insulin) was set up as control. No serum was found in the medium. After 24 hours, the adherent cells were digested with non-enzymatic digestive solution. The adherent cells were prepared into a single cell suspension. After washing with washing buffer, the cells were dissolved in the binding buffer. Every 5 *105 cells and 250 nM aptamer 25 pmole were incubated at 4 C for 30 minutes. The binding degree of aptamers to these cells was analyzed before and after high insulin treatment.
RESULTS: The binding of IR04 to HepG2 cells was inhibited by 100nM insulin, but the binding of IR04 to HepG2 cells was almost completely reduced after 500nM insulin treatment. Similarly, the binding of IR04 to HepG2 cells was not affected by physiological dose of insulin. The binding of IR04 to WT cells was also weakened by hormone treatment, but the binding of IR04 to IRS2/KO cells was not affected by high insulin treatment, while the binding of IR01, IR03 and IR06 to WT cells was not affected by high insulin treatment.
CONCLUSION: The binding of IR04 to target cells is weakened by hyperinsulinemia, and the degree of reduction is positively correlated with the concentration of hyperinsulinemia; however, insulin in the physiological concentration range does not affect the binding of IR04 to target cells; the possible target of IR04 is a membrane protein associated with insulin action on the surface of hepatocytes, which is high in content. Insulin is down-regulated during insulin treatment, but the specific mechanism remains to be further studied; the target substance IR04 is highly homologous to human and mouse hepatocytes, and its mechanism may be similar to human.
The third part is insulin detection based on aptamer modified graphene.
AIM: To detect insulin with aptamer modified graphene oxide (GO) and amplify the detection signal by adding a catalyst (DNA enzyme).
After removing sodium chloride, the grinded graphite powder was added into concentrated sulfuric acid, potassium permanganate was added under strong stirring, and the remaining potassium permanganate and manganese dioxide were reduced by 3% hydrogen peroxide to form colorless and soluble manganese sulfate. After treatment with hydrogen peroxide, the suspension turns bright yellow. Finally, the suspension is filtered and washed three times, and then treated by ultrasonic wave for 4 hours.
An insulin binding aptamer (IBA) labeled with fluorescein isothiocyanate (FITC) was diluted into a 100nM insulin buffer. IBA and GO were mixed at room temperature at a molar concentration of 1:1 to avoid light incubation for 30 minutes. This is the working solution of this experiment. Because GO can quench the fluorescence carried by IBA adsorbed on its surface, at this time the fluorescence can be quenched. There is only a weak fluorescence signal in the solution. When there is insulin in the solution, the IBA bound with GO dissociates from the GO surface, binds with insulin, and the IBA labeled with FITC dissociates into the solution. The quenching effect of GO on FITC is greatly weakened, so that the fluorescence signal in the solution is increased. Different concentrations of insulin (insulin dosage from 5nM to 50mu M) were incubated for 2h. At this time, the insulin-bound IBA was digested into fragments by DNA enzymes, thus losing the ability to bind to insulin, and insulin was re-dissociated in the working fluid; while the GO-bound IBA was protected from digestion by DNA enzymes, but would be dissociated from the GO surface, and In theory, the reaction can be circulated indefinitely until IBA is exhausted. Fluorescence concentration in the solution is detected after termination of the reaction. A negative control group (biotin, streptavidin, bovine serum protein) is set up to determine the specificity of the above insulin detection system.
RESULTS: By modifying the aptamer of insulin nucleic acid on the surface of graphene oxide monolayer, the biosensor of insulin was successfully constructed, and the detection limit of insulin was 500 nM. The detection limit of insulin was reduced to 5 nM by adding DNA enzyme to amplify the signal.
Conclusion: The graphene oxide modified with aptamer insulin nucleic acid can be used as a good tool for insulin detection. The detection limit can be reduced 100 times by adding DNA enzyme to amplify the detection signal.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346
本文編號(hào)：2176884
[Abstract]:The first part is to establish a system of phylogeny for exponential enrichment of hepatocytes.
OBJECTIVE: To establish a ligand system evolution technique (SELEX) for exponential enrichment of hepatocytes, and to obtain an aptamer (Aptamer) for recognizing hepatocyte surface membrane proteins.
METHODS: DNA libraries were designed with 20 BP primer sequences at each end. Primer 1 was labeled with fluorescein isothiocyanate (FITC), primer 2 was labeled with biotin, and the intermediate 20 BP was a random sequence. The cells were washed twice with the washing buffer. The cells were collected with a cell scraper and washed with 1 ml distilled water. The cell suspension was collected in a 1.5 ml centrifugal tube. The supernatant was centrifuged at 95 C for 15 minutes. The mother liquor of the round-screening contains the sequence bound to HepG2. The amplified product is double-stranded DNA containing target and complementary chains. The PCR product is incubated with streptavidin-coated agarose beads and the supernatant is filtered. The double-stranded structure of the PCR is opened with 2% NaOH, and the target chain is dissolved. NaOH was removed from NaOH solution by NAP-5 desalination column. Single-stranded DNA (ssDNA) concentration was determined to determine the total amount of the final screening product and then dried. 100 pmole was extracted from the final screening product and dissolved in a combination buffer containing 10% fetal bovine serum at 500 ul. The growth abundance of the final product in the culture dish (60mm) was 95%. After every 3-5 rounds of screening, we need to check the binding of the products to the cells. When the cell growth abundance reaches about 95%, the cells are digested with non-enzymatic digestive juice. The adherent cells are prepared into a single cell suspension and washed with a washing buffer to dissolve the cells into a combination. In the buffer solution, the screening products of 25 pmole and 5 *105 cells were incubated at 4 C for 30 min and the concentration of screening products was 250 nM. At the same time, the cells were incubated with DNA libraries of the same concentration as negative control. After incubation, the cells were washed and the binding degree between screening products and cells was analyzed by flow cytometry. Three rounds of products with the highest binding degree were selected for sequencing, and the sequence was aptamer for target cells. The DNA synthesized by DNA synthesizer and the binding degree between each sequence and target cells was detected by flow cytometry to determine the final nucleic acid. Aptamers were incubated with trypsin and protease K for 5 min to 30 min, respectively. The aptamers were observed before and after treatment. At the same time, aptamers were incubated with different kinds of cells (including LH86, Huh7, WT, IRS/KO, M7617, Ramos, CEM, H23, H69, A549, HBE, H661, TOV-21G, CAOV3, etc.) and observed. The combination of sequences.
RESULTS: Four aptamers, IR01, IR03, IR04 and IR06, were identified by SELEX. Among them, IR01, IR04 had the best affinity with target cells, and their dissociation constants were 11.287 (+ 3.786nM) and 88.849 (+ 22.339nM), respectively. The dissociation constant was high (129.513 47.924nM), but its specificity was the best, and it could bind to HepG2 cells with specificity and hardly cross-react with other kinds of cells; IR01, IR04, IR06 had good binding to most of the hepatic cell lines; IR01 was the most binding sequence between the aptamer and the target cells. In contrast, the binding of three aptamers IR03, IR04 and IR06 to target cells is easily broken by enzymatic digestion, and the binding of aptamers to target cells is treated by trypsin or protease K. The binding of these three aptamers to target cells was completely destroyed after HepG2 cells 5min.
Conclusion: High affinity and specific binding aptamers with target cells can be obtained by SELEX. These aptamers can identify different cell lines expressing the same biological molecules by recognizing some biological molecules expressed on the target cell membrane.
The second part screened aptamers targeting hepatocyte surface associated with high insulin processing membrane proteins.
AIM: To screen aptamers (Aptamers) targeting the membrane proteins associated with hyperinsulinemia on the surface of hepatocytes.
Methods: After the growth of HepG2 cells was about 70%, the serum was starved for 12 hours, and then divided into four groups. The first group continued to be cultured in insulin-free medium, the second group was cultured in a physiological dose of insulin (0.1nM), the third group was cultured in a high insulin (100nM) medium, and the fourth group was cultured in a super-high insulin (100nM) medium. At the same time, the wild type (WT) and insulin receptor substrate 2 (IRS2) knockout fetal rat hepatocytes (IRS2 / KO) were treated with high insulin (100nM) and the normal culture group (without insulin) was set up as control. No serum was found in the medium. After 24 hours, the adherent cells were digested with non-enzymatic digestive solution. The adherent cells were prepared into a single cell suspension. After washing with washing buffer, the cells were dissolved in the binding buffer. Every 5 *105 cells and 250 nM aptamer 25 pmole were incubated at 4 C for 30 minutes. The binding degree of aptamers to these cells was analyzed before and after high insulin treatment.
RESULTS: The binding of IR04 to HepG2 cells was inhibited by 100nM insulin, but the binding of IR04 to HepG2 cells was almost completely reduced after 500nM insulin treatment. Similarly, the binding of IR04 to HepG2 cells was not affected by physiological dose of insulin. The binding of IR04 to WT cells was also weakened by hormone treatment, but the binding of IR04 to IRS2/KO cells was not affected by high insulin treatment, while the binding of IR01, IR03 and IR06 to WT cells was not affected by high insulin treatment.
CONCLUSION: The binding of IR04 to target cells is weakened by hyperinsulinemia, and the degree of reduction is positively correlated with the concentration of hyperinsulinemia; however, insulin in the physiological concentration range does not affect the binding of IR04 to target cells; the possible target of IR04 is a membrane protein associated with insulin action on the surface of hepatocytes, which is high in content. Insulin is down-regulated during insulin treatment, but the specific mechanism remains to be further studied; the target substance IR04 is highly homologous to human and mouse hepatocytes, and its mechanism may be similar to human.
The third part is insulin detection based on aptamer modified graphene.
AIM: To detect insulin with aptamer modified graphene oxide (GO) and amplify the detection signal by adding a catalyst (DNA enzyme).
After removing sodium chloride, the grinded graphite powder was added into concentrated sulfuric acid, potassium permanganate was added under strong stirring, and the remaining potassium permanganate and manganese dioxide were reduced by 3% hydrogen peroxide to form colorless and soluble manganese sulfate. After treatment with hydrogen peroxide, the suspension turns bright yellow. Finally, the suspension is filtered and washed three times, and then treated by ultrasonic wave for 4 hours.
An insulin binding aptamer (IBA) labeled with fluorescein isothiocyanate (FITC) was diluted into a 100nM insulin buffer. IBA and GO were mixed at room temperature at a molar concentration of 1:1 to avoid light incubation for 30 minutes. This is the working solution of this experiment. Because GO can quench the fluorescence carried by IBA adsorbed on its surface, at this time the fluorescence can be quenched. There is only a weak fluorescence signal in the solution. When there is insulin in the solution, the IBA bound with GO dissociates from the GO surface, binds with insulin, and the IBA labeled with FITC dissociates into the solution. The quenching effect of GO on FITC is greatly weakened, so that the fluorescence signal in the solution is increased. Different concentrations of insulin (insulin dosage from 5nM to 50mu M) were incubated for 2h. At this time, the insulin-bound IBA was digested into fragments by DNA enzymes, thus losing the ability to bind to insulin, and insulin was re-dissociated in the working fluid; while the GO-bound IBA was protected from digestion by DNA enzymes, but would be dissociated from the GO surface, and In theory, the reaction can be circulated indefinitely until IBA is exhausted. Fluorescence concentration in the solution is detected after termination of the reaction. A negative control group (biotin, streptavidin, bovine serum protein) is set up to determine the specificity of the above insulin detection system.
RESULTS: By modifying the aptamer of insulin nucleic acid on the surface of graphene oxide monolayer, the biosensor of insulin was successfully constructed, and the detection limit of insulin was 500 nM. The detection limit of insulin was reduced to 5 nM by adding DNA enzyme to amplify the signal.
Conclusion: The graphene oxide modified with aptamer insulin nucleic acid can be used as a good tool for insulin detection. The detection limit can be reduced 100 times by adding DNA enzyme to amplify the detection signal.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 陳俊意;黃愛(ài)龍;徐莉;陳典全;余虹;朱照靜;黃祖春;楊宗發(fā);陳立書;譚濤;;HBV亞基因型B和C體外重組中間體的檢測(cè)(英文)[J];中南大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2011年02期
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