siRNA干擾ffh、htrA基因?qū)ψ冃捂溓蚓头甓玖σ蜃拥挠绊?/H1>
發(fā)布時(shí)間：2018-05-12 08:43
本文選題：siRNA + htrA基因�。� 參考：《吉林大學(xué)》2014年碩士論文
【摘要】：目的： 齲病是一種非常常見的口腔疾病，是由多種因素共同作用所導(dǎo)致的牙齒硬組織的進(jìn)行性病損。其中細(xì)菌感染是齲病發(fā)生的重要因素之一[1.2.3]，尤其多發(fā)于兒童[4]。在對(duì)齲病微生物學(xué)特性研究結(jié)果顯示，變形鏈球菌[5]是致齲能力最強(qiáng)的齲病致病菌[6-7]。耐酸性和產(chǎn)酸性是變形鏈球菌在口腔環(huán)境中尤其是牙的生物膜上生存的一個(gè)重要因素[8]。研究[3]表明：ffh基因的表達(dá)和htrA基因的表達(dá)與變形鏈球菌的產(chǎn)酸，耐酸及生物膜的形成密切相關(guān)。本研究通過對(duì)比siRNA[9]靶向分別沉默ffh基因[10-11]和htrA[12]基因后變形鏈球菌在強(qiáng)酸弱酸及各種糖的作用下，研究其耐氟菌株的產(chǎn)酸性、耐酸性及生物膜[13]的形成等生物學(xué)性狀的影響。更深刻的揭示變形鏈球菌的耐酸機(jī)制，也為齲病的防治提供新的思路和理論指導(dǎo)。 方法： 體外誘導(dǎo)變形鏈球菌耐氟菌株根據(jù)本實(shí)驗(yàn)組常規(guī)方法將鑒定后的變形鏈球菌放入以50ug/ml NaF遞加直到在還有1000ug/ml NaF的BHI固體培養(yǎng)基中獲得耐氟菌落。 電穿孔轉(zhuǎn)染將鑒定后的UA159-FR懸液與siRNA oligo按2:1比例混合加入電轉(zhuǎn)杯中電極選擇適合電極。用real time-PCR方法檢測(cè)轉(zhuǎn)染效果并篩選出條件較好的siRNA序列。 功能檢測(cè)將轉(zhuǎn)染后的UA159-FR培養(yǎng)在以0.5為間隔，配置pH從3.5至7.0的培養(yǎng)液中進(jìn)行耐酸檢測(cè)收集數(shù)據(jù)并分析；培養(yǎng)在含有5%不同糖類的BHI培養(yǎng)液中進(jìn)行產(chǎn)酸檢測(cè)收集數(shù)據(jù)并分析；培養(yǎng)在放有蓋玻片[14]的孔板中觀察生物膜的形成。 結(jié)果： siRNA oligo在變形鏈球菌耐氟菌株中成功干擾ffh基因和htrA基因表達(dá)。應(yīng)用real-time PCR技術(shù)對(duì)四組siRNA序列進(jìn)行篩選，得出干擾較好的一組siRNA序列進(jìn)行后續(xù)的實(shí)驗(yàn)。 對(duì)干擾后的UA159-FR進(jìn)行產(chǎn)耐酸檢測(cè)，干擾htrA基因的數(shù)據(jù)優(yōu)于干擾ffh基因的數(shù)據(jù)。證實(shí)siRNA對(duì)變形鏈球菌的干擾是有效的。檢測(cè)效果時(shí)間均規(guī)定為24小時(shí)以便對(duì)比數(shù)據(jù)及分析。 在生物膜構(gòu)建過程中，干擾htrA基因的UA159-FR沒能觀察到完整的類似生物膜結(jié)構(gòu)；干擾ffh基因的UA159-FR可觀察到類似生物膜結(jié)構(gòu)，，但是結(jié)構(gòu)上有大小不等的孔隙。 結(jié)論： 成功將siRNA序列電穿孔轉(zhuǎn)染進(jìn)入變形鏈球菌耐氟菌株當(dāng)中，成功分別干擾變形鏈球菌耐氟菌株的ffh基因和htrA基因的表達(dá)，并成功檢測(cè)干擾后功能，得出產(chǎn)酸耐酸和生物膜的樣本及相關(guān)檢測(cè)數(shù)據(jù)并分析。
[Abstract]:Objective: Dental caries is a very common oral disease. It is a progressive lesion of hard tissue caused by various factors. Bacterial infection is one of the important factors in the occurrence of caries [1.2.3], especially in children [4]. The microbiological characteristics of dental caries showed that Streptococcus mutans [5] was the most potent cariogenic pathogen [6-7]. Acid tolerance and acidogenesis are important factors for the survival of Streptococcus mutans in oral environment, especially on dental biofilm [8]. The study [3] showed that the expression of the htrA gene and the expression of the htrA gene were closely related to the production of acid, acid tolerance and biofilm formation of Streptococcus mutans. In this study, we compared the effects of siRNA [9] targeted silencing ffh gene [10-11] and htrA [12] gene on the production of acid, acid tolerance and biofilm formation of fluorine-tolerant strains under the action of strong acid and weak acid and various sugars. The mechanism of acid tolerance of Streptococcus mutans is revealed more deeply, and it also provides new ideas and theoretical guidance for the prevention and treatment of caries. Methods: Fluoride-resistant strains of Streptococcus mutans were induced in vitro. The identified Streptococcus mutans were added with 50ug/ml NaF until they were obtained in BHI solid medium with 1000ug/ml NaF. Electroporation transfection mixed the identified UA159-FR suspension and siRNA oligo at 2:1 ratio to the electrode in the electrorotary cup to select the suitable electrode. The transfection effect was detected by real time-PCR method and the siRNA sequence with better conditions was screened out. After transfection, the UA159-FR were cultured at 0.5 intervals and the pH ranged from 3.5 to 7.0. The acid-resistance data were collected and analyzed in the culture medium containing 5% different sugars, and the acid-producing data were collected and analyzed in the culture medium containing 5% different sugars. The biofilm formation is observed in a hole plate with a cover glass [14]. Results: SiRNA oligo successfully interfered with the expression of ffh and htrA genes in Streptococcus mutans. Four groups of siRNA sequences were screened by real-time PCR technique, and a group of siRNA sequences with good interference was obtained for subsequent experiments. The data of interfering htrA gene was better than that of interfering ffh gene. It is proved that siRNA is effective in interfering with Streptococcus mutans. The effect time is 24 hours to compare data and analysis. During the process of biofilm construction, UA159-FR which interfered with htrA gene could not observe the complete biofilm-like structure, while UA159-FR which interfered with ffh gene could observe biofilm-like structure, but there were some holes in the structure. Conclusion: The siRNA sequence was successfully transfected into Streptococcus mutans fluoride resistant strain, and the expression of ffh gene and htrA gene were interfered respectively, and the function after interference was detected successfully. Acid-resistant and biofilm samples were obtained and analyzed.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R781.1
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 劉莉;張志民;王麗穎;鄭鵬;李會(huì);;變形鏈球菌耐氟菌株中耐酸相關(guān)基因gluA突變的檢測(cè)及其意義[J];吉林大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2011年01期
2 姜葳;梁景平;;變形鏈球菌耐酸反應(yīng)機(jī)制的研究進(jìn)展[J];國際口腔醫(yī)學(xué)雜志;2007年01期
3 Wei Guo;Wangbing Chen;Wendan Yu;Wenlin Huang;Wuguo Deng;;Small interfering RNA-based molecular therapy of cancers[J];Chinese Journal of Cancer;2013年09期
4 Sobia Idrees;Usman A Ashfaq;;RNAi:antiviral therapy against dengue virus[J];Asian Pacific Journal of Tropical Biomedicine;2013年03期
5 姜葳;梁景平;李超倫;姜云濤;;遠(yuǎn)緣鏈球菌6715及其耐氟菌株適應(yīng)性耐酸能力的比較研究[J];口腔醫(yī)學(xué)研究;2007年01期
6 李偉明;石四箴;;澳門幼兒乳牙齲病及齲病活躍性的分析研究[J];臨床口腔醫(yī)學(xué)雜志;2010年05期
7 許世梃;石四箴;梁勤;;兩種齲病活躍性檢測(cè)法的比較研究[J];上海醫(yī)學(xué);2010年07期
8 曹元書;劉興容;;不同齲敏感兒童變形鏈球菌臨床分離株產(chǎn)酸、耐酸能力的實(shí)驗(yàn)研究[J];實(shí)用口腔醫(yī)學(xué)雜志;2009年01期
9 朱敏;張志民;董倩;葛久禹;王文梅;;血鏈球菌和變形鏈球菌耐酸相關(guān)基因ffh的同源性分析[J];實(shí)用老年醫(yī)學(xué);2010年01期
10 米媛;于丹妮;韓育植;張耀超;;變形鏈球菌LuxS基因缺陷株生物膜結(jié)構(gòu)的初步觀察[J];天津醫(yī)藥;2009年07期
本文編號(hào)：1877932
本文鏈接：http://sikaile.net/yixuelunwen/kouq/1877932.html
本文選題：siRNA + htrA基因�。� 參考：《吉林大學(xué)》2014年碩士論文
【摘要】：目的： 齲病是一種非常常見的口腔疾病，是由多種因素共同作用所導(dǎo)致的牙齒硬組織的進(jìn)行性病損。其中細(xì)菌感染是齲病發(fā)生的重要因素之一[1.2.3]，尤其多發(fā)于兒童[4]。在對(duì)齲病微生物學(xué)特性研究結(jié)果顯示，變形鏈球菌[5]是致齲能力最強(qiáng)的齲病致病菌[6-7]。耐酸性和產(chǎn)酸性是變形鏈球菌在口腔環(huán)境中尤其是牙的生物膜上生存的一個(gè)重要因素[8]。研究[3]表明：ffh基因的表達(dá)和htrA基因的表達(dá)與變形鏈球菌的產(chǎn)酸，耐酸及生物膜的形成密切相關(guān)。本研究通過對(duì)比siRNA[9]靶向分別沉默ffh基因[10-11]和htrA[12]基因后變形鏈球菌在強(qiáng)酸弱酸及各種糖的作用下，研究其耐氟菌株的產(chǎn)酸性、耐酸性及生物膜[13]的形成等生物學(xué)性狀的影響。更深刻的揭示變形鏈球菌的耐酸機(jī)制，也為齲病的防治提供新的思路和理論指導(dǎo)。 方法： 體外誘導(dǎo)變形鏈球菌耐氟菌株根據(jù)本實(shí)驗(yàn)組常規(guī)方法將鑒定后的變形鏈球菌放入以50ug/ml NaF遞加直到在還有1000ug/ml NaF的BHI固體培養(yǎng)基中獲得耐氟菌落。 電穿孔轉(zhuǎn)染將鑒定后的UA159-FR懸液與siRNA oligo按2:1比例混合加入電轉(zhuǎn)杯中電極選擇適合電極。用real time-PCR方法檢測(cè)轉(zhuǎn)染效果并篩選出條件較好的siRNA序列。 功能檢測(cè)將轉(zhuǎn)染后的UA159-FR培養(yǎng)在以0.5為間隔，配置pH從3.5至7.0的培養(yǎng)液中進(jìn)行耐酸檢測(cè)收集數(shù)據(jù)并分析；培養(yǎng)在含有5%不同糖類的BHI培養(yǎng)液中進(jìn)行產(chǎn)酸檢測(cè)收集數(shù)據(jù)并分析；培養(yǎng)在放有蓋玻片[14]的孔板中觀察生物膜的形成。 結(jié)果： siRNA oligo在變形鏈球菌耐氟菌株中成功干擾ffh基因和htrA基因表達(dá)。應(yīng)用real-time PCR技術(shù)對(duì)四組siRNA序列進(jìn)行篩選，得出干擾較好的一組siRNA序列進(jìn)行后續(xù)的實(shí)驗(yàn)。 對(duì)干擾后的UA159-FR進(jìn)行產(chǎn)耐酸檢測(cè)，干擾htrA基因的數(shù)據(jù)優(yōu)于干擾ffh基因的數(shù)據(jù)。證實(shí)siRNA對(duì)變形鏈球菌的干擾是有效的。檢測(cè)效果時(shí)間均規(guī)定為24小時(shí)以便對(duì)比數(shù)據(jù)及分析。 在生物膜構(gòu)建過程中，干擾htrA基因的UA159-FR沒能觀察到完整的類似生物膜結(jié)構(gòu)；干擾ffh基因的UA159-FR可觀察到類似生物膜結(jié)構(gòu)，，但是結(jié)構(gòu)上有大小不等的孔隙。 結(jié)論： 成功將siRNA序列電穿孔轉(zhuǎn)染進(jìn)入變形鏈球菌耐氟菌株當(dāng)中，成功分別干擾變形鏈球菌耐氟菌株的ffh基因和htrA基因的表達(dá)，并成功檢測(cè)干擾后功能，得出產(chǎn)酸耐酸和生物膜的樣本及相關(guān)檢測(cè)數(shù)據(jù)并分析。
[Abstract]:Objective: Dental caries is a very common oral disease. It is a progressive lesion of hard tissue caused by various factors. Bacterial infection is one of the important factors in the occurrence of caries [1.2.3], especially in children [4]. The microbiological characteristics of dental caries showed that Streptococcus mutans [5] was the most potent cariogenic pathogen [6-7]. Acid tolerance and acidogenesis are important factors for the survival of Streptococcus mutans in oral environment, especially on dental biofilm [8]. The study [3] showed that the expression of the htrA gene and the expression of the htrA gene were closely related to the production of acid, acid tolerance and biofilm formation of Streptococcus mutans. In this study, we compared the effects of siRNA [9] targeted silencing ffh gene [10-11] and htrA [12] gene on the production of acid, acid tolerance and biofilm formation of fluorine-tolerant strains under the action of strong acid and weak acid and various sugars. The mechanism of acid tolerance of Streptococcus mutans is revealed more deeply, and it also provides new ideas and theoretical guidance for the prevention and treatment of caries. Methods: Fluoride-resistant strains of Streptococcus mutans were induced in vitro. The identified Streptococcus mutans were added with 50ug/ml NaF until they were obtained in BHI solid medium with 1000ug/ml NaF. Electroporation transfection mixed the identified UA159-FR suspension and siRNA oligo at 2:1 ratio to the electrode in the electrorotary cup to select the suitable electrode. The transfection effect was detected by real time-PCR method and the siRNA sequence with better conditions was screened out. After transfection, the UA159-FR were cultured at 0.5 intervals and the pH ranged from 3.5 to 7.0. The acid-resistance data were collected and analyzed in the culture medium containing 5% different sugars, and the acid-producing data were collected and analyzed in the culture medium containing 5% different sugars. The biofilm formation is observed in a hole plate with a cover glass [14]. Results: SiRNA oligo successfully interfered with the expression of ffh and htrA genes in Streptococcus mutans. Four groups of siRNA sequences were screened by real-time PCR technique, and a group of siRNA sequences with good interference was obtained for subsequent experiments. The data of interfering htrA gene was better than that of interfering ffh gene. It is proved that siRNA is effective in interfering with Streptococcus mutans. The effect time is 24 hours to compare data and analysis. During the process of biofilm construction, UA159-FR which interfered with htrA gene could not observe the complete biofilm-like structure, while UA159-FR which interfered with ffh gene could observe biofilm-like structure, but there were some holes in the structure. Conclusion: The siRNA sequence was successfully transfected into Streptococcus mutans fluoride resistant strain, and the expression of ffh gene and htrA gene were interfered respectively, and the function after interference was detected successfully. Acid-resistant and biofilm samples were obtained and analyzed.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R781.1
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 劉莉;張志民;王麗穎;鄭鵬;李會(huì);;變形鏈球菌耐氟菌株中耐酸相關(guān)基因gluA突變的檢測(cè)及其意義[J];吉林大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2011年01期
2 姜葳;梁景平;;變形鏈球菌耐酸反應(yīng)機(jī)制的研究進(jìn)展[J];國際口腔醫(yī)學(xué)雜志;2007年01期
3 Wei Guo;Wangbing Chen;Wendan Yu;Wenlin Huang;Wuguo Deng;;Small interfering RNA-based molecular therapy of cancers[J];Chinese Journal of Cancer;2013年09期
4 Sobia Idrees;Usman A Ashfaq;;RNAi:antiviral therapy against dengue virus[J];Asian Pacific Journal of Tropical Biomedicine;2013年03期
5 姜葳;梁景平;李超倫;姜云濤;;遠(yuǎn)緣鏈球菌6715及其耐氟菌株適應(yīng)性耐酸能力的比較研究[J];口腔醫(yī)學(xué)研究;2007年01期
6 李偉明;石四箴;;澳門幼兒乳牙齲病及齲病活躍性的分析研究[J];臨床口腔醫(yī)學(xué)雜志;2010年05期
7 許世梃;石四箴;梁勤;;兩種齲病活躍性檢測(cè)法的比較研究[J];上海醫(yī)學(xué);2010年07期
8 曹元書;劉興容;;不同齲敏感兒童變形鏈球菌臨床分離株產(chǎn)酸、耐酸能力的實(shí)驗(yàn)研究[J];實(shí)用口腔醫(yī)學(xué)雜志;2009年01期
9 朱敏;張志民;董倩;葛久禹;王文梅;;血鏈球菌和變形鏈球菌耐酸相關(guān)基因ffh的同源性分析[J];實(shí)用老年醫(yī)學(xué);2010年01期
10 米媛;于丹妮;韓育植;張耀超;;變形鏈球菌LuxS基因缺陷株生物膜結(jié)構(gòu)的初步觀察[J];天津醫(yī)藥;2009年07期
本文編號(hào)：1877932
本文鏈接：http://sikaile.net/yixuelunwen/kouq/1877932.html
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