發(fā)布時(shí)間：2018-03-17 17:40

  本文選題：納米細(xì)菌　切入點(diǎn)：納米細(xì)菌檢測(cè)　出處：《華北煤炭醫(yī)學(xué)院》2008年碩士論文　論文類型：學(xué)位論文

【摘要】：納米細(xì)菌(Nanobacteria,Nb)是由芬蘭科學(xué)家Kajander于1989年發(fā)現(xiàn)的一種超微細(xì)菌,它們呈球形或桿狀,具有細(xì)胞壁,直徑在80～500nm之間,菌落常呈蔟狀;能生成含有磷灰石的生物膜,具有很強(qiáng)的抗熱、抗γ射線輻射及抵御抗生素的能力;能通過100nm的濾菌器,且具獨(dú)特的生物礦化能力。近年來,有研究資料表明,納米細(xì)菌與膽囊結(jié)石、腎結(jié)石、動(dòng)脈粥樣硬化等多種骨骼外鈣化性或硬化性疾病有關(guān),引起了科學(xué)界極大的關(guān)注。 采用抗納米細(xì)菌單克隆抗體作免疫熒光及免疫組化檢測(cè),掃描電鏡、透射電鏡及免疫電鏡等形態(tài)學(xué)觀察,是目前鑒定納米細(xì)菌的主要方法。 目的 本研究旨在通過對(duì)膽石癥患者膽汁納米細(xì)菌分離培養(yǎng),免疫動(dòng)物,制備多克隆抗體;并對(duì)試驗(yàn)條件的探討優(yōu)選,建立斑點(diǎn)酶免疫滲濾法檢測(cè)動(dòng)物血清中特異性納米細(xì)菌抗體的初步診斷方法。 方法 1.選擇無急性膽囊炎發(fā)作病史,腹腔鏡手術(shù)前未接受抗生素治療的6例膽囊結(jié)石患者,無菌操作抽取膽汁進(jìn)行納米細(xì)菌培養(yǎng),同時(shí)設(shè)DMEM全培養(yǎng)液培養(yǎng)和合成羥基磷灰石培養(yǎng)為陰性對(duì)照。 2.用數(shù)顯濁度儀測(cè)量膽石癥患者膽汁分離培養(yǎng)物和對(duì)照組中納米細(xì)菌生長(zhǎng)情況并繪制生長(zhǎng)曲線。 3.用掃描電鏡對(duì)膽石癥患者膽汁分離培養(yǎng)物進(jìn)行能譜分析,并用透射電鏡對(duì)納米細(xì)菌培養(yǎng)物進(jìn)行形態(tài)學(xué)觀察。 4.用納米細(xì)菌培養(yǎng)物制成納米細(xì)菌滴片,分別按文獻(xiàn)方法進(jìn)行間接免疫熒光染色、Von KOSSA染色及Hochest 33258染色。 5.純化抗原,通過考馬斯亮藍(lán)定量蛋白濃度為174.2μg/ml,保存?zhèn)溆谩?6.免疫家兔 免疫家兔3只,背部多點(diǎn)接種含福氏完全佐劑的納米細(xì)菌(總量200μg/ml/只)。2周后接種含福氏不完全佐劑的納米細(xì)菌(總量50μg/ml/只)進(jìn)行加強(qiáng)免疫(每次間隔2周,共4次),制備納米細(xì)菌多克隆抗體,并用ELISA測(cè)定其含量。 7.建立納米細(xì)菌多克隆抗體斑點(diǎn)酶免疫滲濾檢測(cè)法 將兔抗納米細(xì)菌多克隆抗體用二倍順序稀釋法稀釋至1:6400～1:51200;納米細(xì)菌抗原稀釋至1?10～1:160等不同濃度;通用型抗鼠/兔酶標(biāo)抗體稀釋至1:10～1:40。用方陣滴定法確定所有試驗(yàn)點(diǎn)及其相應(yīng)的抗原抗體反應(yīng)濃度。每個(gè)試驗(yàn)點(diǎn)重復(fù)6次,以出現(xiàn)肉眼能判讀的棕色斑點(diǎn)或圓圈者為陽性,不能判讀者為陰性。對(duì)每個(gè)陽性反應(yīng)棕色斑點(diǎn),用“Motic Med 6.0數(shù)碼醫(yī)學(xué)圖象分析系統(tǒng)(A)”讀取光密度值(OD值),求出每個(gè)試驗(yàn)點(diǎn)的平均OD值。 分析數(shù)據(jù),選擇檢測(cè)納米細(xì)菌多抗的最佳兔抗納米細(xì)菌抗原滴度和酶標(biāo)抗體滴度的反應(yīng)組合�？乖兔笜�(biāo)抗體滴度固定后,進(jìn)一步檢測(cè)不同稀釋度的多克隆抗體,讀取數(shù)據(jù),分析OD值變化與納米細(xì)菌多抗含量的關(guān)系,繪制標(biāo)準(zhǔn)曲線,求出回歸方程及相關(guān)系數(shù)。 8.斑點(diǎn)酶免疫滲濾法的特異性檢測(cè)及評(píng)價(jià) 用最佳納米細(xì)菌抗原滴度和酶標(biāo)抗體滴度的反應(yīng)組合,以斑點(diǎn)酶免疫滲濾法對(duì)不同濃度的實(shí)驗(yàn)動(dòng)物感染陽性血清標(biāo)本和陰性血清標(biāo)本進(jìn)行檢測(cè),通過診斷靈敏度、診斷特異性和假陽性率等指標(biāo)確定樣本的最佳稀釋濃度。 用最佳納米細(xì)菌抗原滴度和酶標(biāo)抗體滴度的反應(yīng)組合,對(duì)納米細(xì)菌感染實(shí)驗(yàn)各組家兔血清標(biāo)本進(jìn)行檢測(cè),并評(píng)價(jià)其診斷靈敏度、診斷特異度、陽性結(jié)果預(yù)期值、陰性結(jié)果預(yù)期值及總有效率。 結(jié)果 1. 6例膽石癥患者膽汁樣本在含有10%γ-FBS的DMEM、37℃和5%CO2培養(yǎng)條件下,納米細(xì)菌培養(yǎng)均為陽性。 2.膽石癥患者膽汁培養(yǎng)物在倒置相差顯微鏡下可見做布朗運(yùn)動(dòng)的微小的顆粒。培養(yǎng)過程中納米細(xì)菌緩慢生長(zhǎng),培養(yǎng)液的pH值無明顯變化;傳代后的納米細(xì)菌仍維持上述生物學(xué)特性。 3.經(jīng)數(shù)顯濁度儀測(cè)量,在4周內(nèi),每5天測(cè)量一次,納米細(xì)菌培養(yǎng)組濁度一直呈上升趨勢(shì),其倍增時(shí)間約為3天,而DMEM和羥基磷灰石對(duì)照組無明顯變化,經(jīng)180℃干烤4h滅活的納米細(xì)菌培養(yǎng)組也未見明顯變化。 4.納米細(xì)菌的鑒定 透射電鏡下觀察,納米細(xì)菌約為80～350nm,呈橢球形或短棒狀顆粒,聚集成簇狀,其表面覆有細(xì)菌被膜。 掃描電鏡能譜分析(EDX)顯示,納米細(xì)菌含有鈣、磷、鋁、硅、硫等元素,其鈣/磷比值為1.62,與羥基磷灰石中鈣/磷比值的1.66相近。 Von KOSSA鈣染色法結(jié)果顯示納米細(xì)菌形成的礦化外殼呈黑色。 間接免疫熒光染色顯示納米細(xì)菌被熒光抗體結(jié)合,在熒光顯微鏡下激發(fā)綠色熒光。 納米細(xì)菌可被Hoechst 33258染色,發(fā)出特征性的藍(lán)色熒光,提示納米細(xì)菌含有DNA成分。 5.免疫家兔血清經(jīng)ELISA檢測(cè),多抗的濃度為56.25mg/ml。 6.斑點(diǎn)酶免疫滲濾法檢測(cè)Nb多克隆抗體的最佳反應(yīng)滴度為:Nb抗原1:20(8.71μg/ml),通用型酶標(biāo)抗體濃度1:10。建立不同稀釋濃度的多抗與OD值變化關(guān)系的標(biāo)準(zhǔn)曲線,求出回歸方程Y=0.0832X+0.0745,相關(guān)系數(shù)r=0.876(P0.05)。確定最小Nb多克隆抗體稀釋比例為1:12800,最小抗體檢出濃度為68.59ng/ml。 7.用最佳納米細(xì)菌抗原滴度和酶標(biāo)抗體滴度的反應(yīng)組合,以斑點(diǎn)酶免疫滲濾法對(duì)不同濃度的實(shí)驗(yàn)動(dòng)物感染陽性血清標(biāo)本和陰性血清標(biāo)本進(jìn)行檢測(cè),確定最佳血清樣本工作滴度為1:6400。 8.用斑點(diǎn)酶免疫滲濾法檢測(cè)對(duì)納米細(xì)菌感染實(shí)驗(yàn)各組家兔血清標(biāo)本進(jìn)行檢測(cè)的結(jié)果:40份陽性,56份陰性;而芬蘭nanobac公司ELISA試劑盒檢測(cè)結(jié)果為:38份陽性,58份陰性。檢測(cè)結(jié)果經(jīng)配對(duì)資料χ2檢驗(yàn),與納米細(xì)菌感染家兔致膽結(jié)石結(jié)果相關(guān)(χ2=88.055, P=0.001),且兩者具有高度診斷一致性(kappa值為0.957)。 結(jié)論 1.患有膽囊結(jié)石的患者膽汁中存在納米細(xì)菌感染。納米細(xì)菌體積微小,生長(zhǎng)緩慢,在生理?xiàng)l件下其菌體表面可生成羥基磷灰石礦化外殼。 2.納米細(xì)菌具有免疫原性,可以通過免疫動(dòng)物制備抗血清。 3.初步建立斑點(diǎn)酶免疫滲濾法對(duì)兔血清中納米細(xì)菌抗體檢測(cè)法,其靈敏度:96.55%;特異度:82.09%;約登指數(shù):0.7867;總有效率:86.46%;陽性預(yù)期值:70.00%;陰性預(yù)期值:98.21%。
[Abstract]:Nanobacteria (Nanobacteria, Nb) is a kind of micro bacteria discovered by scientists in Finland Kajander in 1989, they are spherical or rod-shaped, with cell wall, with a diameter of 80 ~ 500nm, colonies are usually tufted; can produce biofilm containing apatite, with strong heat resistance, anti radiation ability and resist antibiotics; through the filter of 100nm, and has the unique ability of biomineralization. In recent years, studies show that with gallstones, kidney stones, atherosclerosis and other bone calcification or sclerosis disease, have attracted great attention.
Immunofluorescence and immunohistochemistry were used for the detection of monoclonal antibodies against nanomaterials. Scanning electron microscopy, transmission electron microscopy and immunoelectron microscopy were the main methods to identify nanomaterials.
objective
The aim of this study is to prepare polyclonal antibodies from bile bacteria and nanomaterials in patients with cholelithiasis.
Method
1., 6 patients without cholecystolithiasis who had no history of acute cholecystitis before laparoscopic surgery were selected. The bacteria were cultured under sterile operation and cultured in nanomaterials. At the same time, DMEM total culture medium and hydroxyapatite culture were used as negative control.
2. the growth of bile separation culture in cholelithiasis patients and the nanoscale in the control group were measured with the digital turbidimeter, and the growth curve was plotted.
3. scanning electron microscopy was used to analyze the bile separation culture of cholelithiasis, and the morphology of the nanoscale bacteria was observed by transmission electron microscope.
4. nanoscale bacterial cultures were used to produce nanoscale bacterial drops. Indirect immunofluorescence staining, Von KOSSA staining and Hochest 33258 staining were carried out according to the literature method.
The 5. purified antigen, and preserved through the examination of quantitative protein concentration of Coomassie blue is 174.2 g/ml.
6. immunized rabbits
Immune 3 rabbits back multipoint inoculation with complete Freund's adjuvant nanobacteria (total 200 g/ml/).2 weeks after inoculation with Freund's incomplete adjuvant nanobacteria (total 50 g/ml/ only) to strengthen immunity (the time interval of 2 weeks, a total of 4 times), the preparation of nanobacteria polyclonal antibody, and its content was determined by ELISA.
7. establishment of multi clone antibody dot enzyme immunofiltration assay for nanoscale bacteria
Rabbit anti nanobacteria polyclonal antibody with two times dilution in order to 1:6400 ~ 1:51200; nanobacterial antigen diluted to 1? 10 ~ 1:160 in different concentration; universal anti mouse / rabbit HRP was diluted to 1:10 ~ 1:40. with square titration method to determine all the test points and the corresponding concentration of the antigen antibody reaction. Each test was repeated 6 times, with the naked eye can interpret brown spots or circle is positive, not negative. For each sentence readers positive brown spots, analysis system with Motic Med 6 digital medical image (A) "to read the value of optical density (OD), calculated the average OD value of each test point.
Analysis of the data, select the best detection of nanobacteria polyclonal anti rabbit nanobacterial antigen titer and the antibody titer of the enzyme labeled antigen and enzyme reaction. Antibody titer after fixation, further detection of polyclonal antibody, different dilutions of the read data, analysis of the relationship between od changes and nano bacteria antibody content, standard curve for the regression equation and correlation coefficient.
Specific detection and evaluation of 8. dot enzyme immunoassay
The reaction in combination with the best nanobacterial antigen titer and the antibody titer by ELISA, dot immuno enzyme filtration assay to detect different concentrations of experimental animal infected with positive serum samples and negative serum samples, the diagnostic sensitivity, specificity and the optimum concentrations of false positive rate and other indicators to determine the sample.
Using the best combination of nano bacterial antigen titer and enzyme antibody titer, we detected the serum samples of each group, and evaluated their diagnostic sensitivity, diagnostic specificity, positive results, expected value and total effective rate.
Result
In 1.6 cases of cholelithiasis, the bile samples were positive in the culture of DMEM, 37 and 5%CO2, with 10% gamma -FBS.
2.. Bile bacteria in cholelithiasis patients can be seen as tiny particles of Brown movement under inverted phase contrast microscope. During the culture process, nanomaterials grew slowly, and the pH value of culture solution did not change significantly.
3. by digital turbidity measurement, in 4 weeks, measured once every 5 days, nanobacteria culture group turbidity has been on the rise, the doubling time is about 3 days, but no obvious changes of DMEM and ha control group cultured groups showed no significant change at 180 dry roasted inactivated 4H nanoparticles bacteria.
Identification of 4. nanoscale bacteria
Under transmission electron microscopy, it was observed that the nanometers were about 80 ~ 350nm, with ellipsoidal or short rod like particles, which were clustered together, and the surface was covered with bacterial membrane.
Scanning electron microscopy (EDX) shows that nanomaterials contain calcium, phosphorus, aluminum, silicon, sulfur and other elements. The ratio of calcium to phosphorus is 1.62, which is close to 1.66 of the ratio of calcium to phosphorus in hydroxyapatite.
The results of Von KOSSA calcium staining showed that the mineralized shell formed by nanometers was black.
Indirect immunofluorescence staining showed that nanophores were combined with fluorescent antibodies and stimulated green fluorescence under the fluorescence microscope.
Nanometers can be stained by Hoechst 33258 and emit a characteristic blue fluorescence, suggesting that nanbacteria contain DNA components.
5.鍏嶇柅瀹跺厰琛，

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