【摘要】：研究背景： 卵巢癌是婦科癌癥死亡的首要原因，晚期卵巢癌的治療主要包括腫瘤細(xì)胞減滅術(shù)和紫杉醇/卡鉑化療。但是大部分患者死于腫瘤復(fù)發(fā)。血管生成是卵巢癌進(jìn)展和轉(zhuǎn)移的基礎(chǔ)，抗血管生成治療是控制和治愈卵巢癌的希望所在。兩個(gè)隨機(jī)研究表明通過添加抗VEGF單克隆抗體貝伐單抗，可延長(zhǎng)無(wú)進(jìn)展生存期（PFS），因此導(dǎo)致FDA批準(zhǔn)貝伐單抗可用卵巢癌的一線治療，也表明靶向血管生成是有前途的抗腫瘤治療策略。 CD105又名Endoglin（EDG），是一種細(xì)胞膜糖蛋白，是內(nèi)皮細(xì)胞增殖的標(biāo)志之一，還是轉(zhuǎn)化生長(zhǎng)因子-β (TGF-β)受體復(fù)合物的組分之一。CD105在腫瘤血管和淋巴管內(nèi)皮細(xì)胞有較高的表達(dá)，而在成熟的血管內(nèi)皮細(xì)胞中表達(dá)較弱或基本不表達(dá)。體外研究表明，內(nèi)皮細(xì)胞對(duì)TGF的反應(yīng)需CD105調(diào)節(jié)，而CD105尚可促內(nèi)皮細(xì)胞增殖，抑制其凋亡，并且有促新生血管生成等功能。大量研究表明，CD105可作為腫瘤顯像及治療的理想靶點(diǎn)。以CD105為標(biāo)記的腫瘤微血管密度（MVD）可判斷多種實(shí)體腫瘤的惡性度及預(yù)后，由MMP-14剪切產(chǎn)生的可溶性的sCD105可用于多種轉(zhuǎn)移性腫瘤，如乳腺癌、腸癌患者的預(yù)后判斷，而多項(xiàng)報(bào)道認(rèn)為子癇前期的孕婦血清中增加sEndoglin水平升高。 卵巢癌是婦科惡性度最高的腫瘤之一，也是血管化程度較高的惡性腫瘤，以貝伐單抗為代表的抗血管生成藥物已顯示出較好的抗腫瘤效應(yīng)，但也存在持續(xù)時(shí)間短、副作用較大等缺點(diǎn)，CD105在卵巢癌微血管內(nèi)皮高表達(dá)，抗人CD105抗體在其診斷及治療中有可能發(fā)揮重要作用。目前有較多有關(guān)CD105單克隆抗體（McAb）或人鼠嵌合抗體及其偶聯(lián)物抗腫瘤的報(bào)道。國(guó)外已有CD105-McAb商品，動(dòng)物試驗(yàn)表明CD105-McAb有較好的抗腫瘤作用，目前人鼠嵌合抗體I期臨床試驗(yàn)正在進(jìn)行，國(guó)內(nèi)尚無(wú)抗人CD105功能性抗體的報(bào)道。研制具有自主知識(shí)產(chǎn)權(quán)的CD105-McAb并用于臨床診斷及治療具有重大的意義。 制備抗人Endoglin抗體不僅能將其應(yīng)用于抗腫瘤藥物的篩選，亦可將其構(gòu)建免疫檢測(cè)技術(shù)應(yīng)用于卵巢癌轉(zhuǎn)移及預(yù)后的評(píng)測(cè)。但是抗體藥物的制備難度大，無(wú)論在特異性、親和力、中和阻斷活性及生物反應(yīng)活性等個(gè)方面均有很高要求。但在診斷應(yīng)用上相對(duì)要求較低，因此在成功制備抗人CD105抗體后，可以充分利用所制備的抗體，建立Endoglin體內(nèi)及體外檢測(cè)技術(shù)。目前檢測(cè)sCD105仍多采用酶聯(lián)免疫吸附法或放射免疫法，均存在檢測(cè)費(fèi)用高，需多人份血清同時(shí)檢測(cè)，等候時(shí)間長(zhǎng)的缺點(diǎn)。電化學(xué)免疫分析法成功的將免疫反應(yīng)的高選擇性和電化學(xué)測(cè)定的高靈敏度相結(jié)合而受到人們的青睞，其測(cè)定靈敏度與放射免疫法相近，而又不需要使用放射性同位素，充分顯示了電化學(xué)免疫法在臨床檢測(cè)中的優(yōu)越性和發(fā)展前途。電化學(xué)免疫分析法自上世紀(jì)70年代提出研究理論至今，已成功的將電化學(xué)檢測(cè)到高靈敏性，高穩(wěn)定性等與免疫反應(yīng)的高選擇性相結(jié)合而研發(fā)出適用于醫(yī)療，，生物等多領(lǐng)域的電化學(xué)免疫傳感器，并越來(lái)越受到人們的青睞，其測(cè)定靈敏度相當(dāng)或優(yōu)于目前高靈敏檢測(cè)所依賴的放射免疫法，而又不需要使用放射性同位素，不僅減少了醫(yī)護(hù)人員及病患的放射損傷，同時(shí)也避免了放射性標(biāo)記對(duì)抗體或抗原等于有標(biāo)記所造成的化學(xué)損傷，而最大程度地保持了抗體或抗原的免疫反應(yīng)活性，提高了檢測(cè)效率。這些都充分顯示了電化學(xué)免疫法在臨床檢測(cè)應(yīng)用中的優(yōu)越性和發(fā)展前景。 主要的研究?jī)?nèi)容和結(jié)果： 1、人CD105分子胞外區(qū)基因的克隆及在原核系統(tǒng)中的表達(dá)與純化： 通過PCR技術(shù)克隆了CD105分子胞外段基因，經(jīng)酶切鑒定及序列分析表明其序列與文獻(xiàn)報(bào)道完全一致。將該片段亞克隆到表達(dá)載體pET32a(+)，轉(zhuǎn)化BL21，經(jīng)IPTG誘導(dǎo)，SD-SAPGE分析，在80kd處有一明顯的誘導(dǎo)表達(dá)帶，其分子量大小與預(yù)期相符。Western-blot分析表明，該表達(dá)蛋白帶可被商業(yè)化CD105-McAb特異識(shí)別。表達(dá)產(chǎn)物以包含涵體形式存在，通過鎳離子柱親和純化得到純化蛋白并復(fù)性，最終獲得純度可達(dá)90%的目的重組蛋白。該蛋白為后續(xù)研究中抗體制備及檢測(cè)技術(shù)建立奠定基礎(chǔ)。 2、人CD105單克隆抗體的制備及鑒定： 2.1CD105表位特異性線性多肽多克隆抗體的制備： 為了能獲得更多不同表位的CD105抗體，達(dá)到建立雙抗體夾心配對(duì)檢測(cè)CD105的目的，采用表位在線分析及文獻(xiàn)報(bào)道CD105抗體的表位，我們?cè)O(shè)計(jì)并合成了CD105分子B細(xì)胞線性表位多肽，并與BSA交聯(lián)。將偶聯(lián)BSA的多肽免疫兔子，得到高滴度的抗體，純化后抗體經(jīng)ELISA、SDS-PAGE等檢測(cè)具有抗CD105特異性。 2.2CD105單克隆抗體的制備及鑒定： 利用純化的CD105胞外段蛋白作為免疫原，我們采用改良的免疫程序，采用快速的雜交瘤技術(shù)，經(jīng)歷4細(xì)胞融合，成功制備了數(shù)十株陽(yáng)性克隆，使用從北京義翹購(gòu)買的CHO細(xì)胞表達(dá)的重組CD105蛋白篩選出6株抗糖基化CD105的特異性單克隆抗體，經(jīng)酶聯(lián)免疫吸附法（ELISA）、Western Blot、細(xì)胞及組織免疫熒光等多種手段對(duì)所得單抗進(jìn)行特異性分析，顯示我們制備的單克隆抗體具有抗CD105特異。 3、131碘(~(131)I)標(biāo)記的CD105單克隆抗體在荷人卵巢癌裸鼠體內(nèi)顯像和分布： 3.1用Iodogen法合成~(131)I-CD105單抗及對(duì)照單抗（鼠抗人單克隆抗體），SephadexG50柱純化，利用紙層析法測(cè)定其標(biāo)記率及放化純度，結(jié)果表明Iodogen法標(biāo)記CD105單抗及對(duì)照單抗，標(biāo)記率分別為92.2%和83.6%，放化純度超過97%，放射比活性分別為147.52μci/μg和129.58μci/μg，放射性濃度達(dá)737.6μci/ml和732.09μc。 3.2經(jīng)鼠尾靜脈注射~(131)I標(biāo)記單抗入荷人卵巢癌裸鼠體內(nèi)，SPECT儀下觀察腫瘤顯像情況，同時(shí)，在注射~(131)I-CD105單抗后24h，48h，72h脫頸椎處死一組小鼠，取各重要臟器及腫瘤組織，濾紙吸干，γ放射免疫計(jì)數(shù)器測(cè)定各組織的每分鐘放射性計(jì)數(shù)，計(jì)算每克組織每分鐘放射性計(jì)數(shù)，計(jì)算放射比活性，計(jì)算腫瘤組織和非腫瘤組織放射性比值（T/NT比值）。SPECT觀察注射后24h，48h，96h放射自顯影成像情況。 實(shí)驗(yàn)組心、肝、肺、腎、胃等組織在24小時(shí)時(shí)點(diǎn)均有較高的每克組織攝取注射量的百分比（%ID/g），隨著時(shí)間的延長(zhǎng)，腫瘤組織的放射性攝取率明顯增高，而其他重要臟器攝取率逐漸下降，至96小時(shí)腫瘤組織每克組織攝取注射量的百分比高達(dá)29.10±1.61%ID/g，而肝、腎等臟器均在明顯下降。相應(yīng)的T/NT比值在96小時(shí)時(shí)點(diǎn)均3，證明~(131)I-CD105單抗能夠在腫瘤組織內(nèi)濃聚并在96小時(shí)達(dá)到高點(diǎn)。而對(duì)照組未發(fā)現(xiàn)腫瘤部位的放射性濃聚。 ~(131)I-CD105單抗組在尾靜脈注射~(131)I標(biāo)記物后，移植瘤部位和腎臟，肝臟，胃開始出現(xiàn)放射性濃聚，隨著時(shí)間的延長(zhǎng)，胸腹部逐漸變淡，腫瘤區(qū)域逐漸濃聚,48小時(shí)組腫瘤區(qū)域清晰可見，96小時(shí)本底背景明顯減弱，而腫瘤顯像明顯增強(qiáng)。 4、人CD105免疫傳感器檢測(cè)的建立及評(píng)測(cè) 在抗原（重組蛋白）及高效抗體制備的基礎(chǔ)上，采用電沉積納米金技術(shù)在電極表面構(gòu)建生物兼容性良好的納米金表面，并進(jìn)一步修飾半胱氨酸用于吸附修飾小粒徑的納米金增加工作面對(duì)比表面積，提高抗體的固定量，將篩選的單克隆抗體固定于制備電極表面，通過抗原/抗體特異性反應(yīng)，以及蛋白在電極表面的結(jié)合對(duì)電化學(xué)響應(yīng)信號(hào)的影響，初步建立了應(yīng)用于CD105檢測(cè)的電化學(xué)免疫傳感器。在此基礎(chǔ)上，為了進(jìn)一步提高免疫檢測(cè)的特異性及靈敏性，采用納米鉑及硫堇（電子媒介體）標(biāo)記的配對(duì)抗CD105抗體作為檢測(cè)抗體，通過硫堇實(shí)現(xiàn)電化學(xué)響應(yīng)，及納米鉑的催化性能進(jìn)一步提高研究的電化學(xué)免疫傳感器的靈敏性，并通過檢測(cè)抗體與所檢測(cè)抗原的特異性反應(yīng)進(jìn)一步提高免疫檢測(cè)體系的特異性，實(shí)現(xiàn)CD105的高特異，高靈敏性檢測(cè)。電化學(xué)免疫傳感器的研究及制備過程，表征鑒定我們采用循環(huán)伏安法（CV）考察了電極表面的電化學(xué)特性，充分地研究了免疫傳感器的性能。采用CV對(duì)人CD105進(jìn)行檢測(cè)，初步研究獲得了良好的檢測(cè)效果，基本建立了應(yīng)用于人CD105檢測(cè)的電化學(xué)免疫傳感器，進(jìn)一步驗(yàn)證了所研究的電化學(xué)免疫傳感技術(shù)的良好實(shí)用性，并充分證明了所研究制備的重組蛋白及高效抗體的良好檢測(cè)應(yīng)用性能及可能的臨床檢測(cè)應(yīng)用前景。同時(shí)，此研究為進(jìn)一步改良并研發(fā)適用于臨床應(yīng)用的CD105檢測(cè)技術(shù)奠定基礎(chǔ)。
[Abstract]:Research background:
Ovarian cancer is the leading cause of death in gynecologic cancer. Treatment for advanced ovarian cancer includes cytoreductive surgery and chemotherapy with paclitaxel/carboplatin. But most patients die of recurrence. Angiogenesis is the basis of ovarian cancer progression and metastasis. Antiangiogenic therapy is the hope of controlling and curing ovarian cancer. Two randomized studies It has been shown that bevacizumab can prolong progression-free survival (PFS) by adding anti-VEGF monoclonal antibody, which leads to FDA approval of bevacizumab as a first-line treatment for ovarian cancer. Targeted angiogenesis is also a promising anti-tumor strategy.
CD105, also known as Endoglin (EDG), is a membrane glycoprotein, a marker of endothelial cell proliferation, or a component of transforming growth factor-beta (TGF-beta) receptor complex. CD105 is highly expressed in tumor blood vessels and lymphatic endothelial cells, but weakly or basically not expressed in mature vascular endothelial cells. These results suggest that the endothelial cell response to TGF is regulated by CD105, which can promote the proliferation of endothelial cells, inhibit their apoptosis and promote angiogenesis. Many studies have shown that CD105 can be used as an ideal target for tumor imaging and treatment. Prognosis, soluble sCD105 produced by MMP-14 shearing can be used to predict the prognosis of many metastatic tumors, such as breast cancer and bowel cancer, and many reports suggest that increased serum sEndoglin levels in preeclampsia pregnant women are elevated.
Ovarian cancer is one of the most malignant tumors in gynecology. It is also a highly vascularized malignant tumor. Bevacizumab as a representative of anti-angiogenesis drugs has shown good anti-tumor effect, but there are also shortcomings such as short duration and side effects. CD105 is highly expressed in the microvascular endothelium of ovarian cancer, and anti-human CD105 antibody in its diagnosis. There are many reports about the anti-tumor effect of CD105 monoclonal antibody (McAb) or human-mouse chimeric antibody and its conjugates. There are CD105-McAb products abroad. Animal tests show that CD105-McAb has a good anti-tumor effect. At present, phase I clinical trials of human-mouse chimeric antibody are under way, but there is no anti-tumor effect in China. It is of great significance to develop CD105-McAb with independent intellectual property rights for clinical diagnosis and treatment.
Preparation of anti-human Endoglin antibody can be used not only in screening of anti-tumor drugs, but also in the detection of metastasis and prognosis of ovarian cancer by constructed immunoassay. Therefore, after the successful preparation of anti-human CD105 antibody, we can make full use of the prepared antibody to establish Endoglin in vivo and in vitro detection technology. Point. Electrochemical immunoassay (EIA) successfully combines the high selectivity of immune reaction with the high sensitivity of electrochemical assay. Its sensitivity is similar to that of radioimmunoassay without the use of radioisotopes. It fully demonstrates the superiority and development prospects of EIA in clinical detection. Since the theory of chemical immunoassay was put forward in the 1970s, electrochemical immunosensors have been successfully developed for medical, biological and other fields by combining electrochemical detection with high sensitivity, high stability and high selectivity of immune reaction, and have attracted more and more attention. It is superior to the radioimmunoassay, which is currently used in highly sensitive detection, and does not require the use of radioisotopes. It not only reduces the radiation damage of medical staff and patients, but also avoids the chemical damage caused by the radiolabeling of antibodies or antigens equal to the labeling, and maintains the immune response of antibodies or antigens to the greatest extent. All these fully demonstrate the superiority and development prospect of electrochemical immunoassay in clinical detection.
Main research contents and results:
1, cloning and expression and purification of the extracellular domain of human CD105 molecule in the prokaryotic system.
The extracellular segment of CD105 gene was cloned by PCR and its sequence was identical with that reported in literatures. The subcloned fragment was transformed into BL21 by subcloning into expression vector pET32a (+). After induction by IPTG and SD-SAPGE analysis, an obvious induction expression band was found at 80 kd. The molecular weight of the fragment was in agreement with the expected value. The results showed that the expressed protein band could be specifically recognized by commercial CD105-McAb. The expressed product was in the form of inclusion body. The purified protein was purified by Ni-ion affinity purification and renatured, and the purity of the recombinant protein was up to 90%. This protein lays a foundation for antibody preparation and detection technology in the follow-up study.
2, preparation and characterization of human CD105 monoclonal antibody.
Preparation of 2.1CD105 epitope specific linear polypeptide polyclonal antibody:
In order to obtain more CD105 antibodies with different epitopes and to establish a double antibody sandwich matching method for detection of CD105, we designed and synthesized a linear epitope polypeptide of CD105 molecule B cells by on-line epitope analysis and reported CD105 antibody epitopes. The polypeptides conjugated with BSA were immunized to rabbits to obtain high titer antibodies. After purification, the antibody was tested for anti CD105 specificity by ELISA and SDS-PAGE.
Preparation and characterization of monoclonal antibodies against 2.2CD105:
Using purified CD105 extracellular segment protein as immunogen, we have successfully prepared dozens of positive clones by rapid hybridoma technique and 4-cell fusion. Six specific monoclonal antibodies against glycosylated CD105 have been screened by using recombinant CD105 protein expressed by CHO cells purchased from Beijing Yiqiao. Enzyme-linked immunosorbent assay (ELISA), Western Blot, cell and tissue immunofluorescence were used to analyze the specificity of the monoclonal antibody. The results showed that the monoclonal antibody prepared by us was specific to CD105.
Imaging and distribution of 3131 iodine (~ (131) I) labeled CD105 monoclonal antibody in nude mice bearing human ovarian cancer:
3.1 The ~ (131) I-CD105 monoclonal antibody and the control monoclonal antibody (mouse anti-Human Monoclonal antibody) were synthesized by Iodogen method and purified by Sephadex G50 column. The labeling rate and radiochemical purity of the monoclonal antibody and the control monoclonal antibody were determined by paper chromatography. The results showed that the labeling rates of the monoclonal antibody and the control monoclonal antibody were 92.2% and 83.6%, respectively. The radiochemical purity of the monoclonal antibody and the control monoclonal antibody were over 97% and 147 / g and 129.58 mu ci/ g, the radioactive concentration is 737.6 ci/ml and 732.09 C.
3.2 Nude mice bearing human ovarian cancer were injected with ~ (131) I-labeled monoclonal antibody via tail vein of mice. The tumor imaging was observed by SPECT. At the same time, a group of mice were sacrificed 24 hours, 48 hours and 72 hours after injection of ~ (131) I-CD105 monoclonal antibody. The vital organs and tumor tissues were taken out, filtered paper was dried, and the minute radioimmunoassay was performed by gamma-ray counter. Radioactive counts per gram of tissue per minute were calculated, radioactivity was calculated, and radioactivity ratios (T/NT ratios) between tumor and non-tumor tissues were calculated. SPECT was used to observe autoradiography 24, 48 and 96 hours after injection.
The heart, liver, lung, kidney, stomach and other tissues in the experimental group all had a higher percentage of uptake of injection per gram of tissue at 24 hours (% ID / g). With the prolongation of time, the uptake rate of radioactivity in tumor tissue increased significantly, while the uptake rate of other important organs gradually decreased, and the percentage of uptake of injection per gram of tumor tissue reached 29.1% at 96 hours. The corresponding T/NT ratios were 3 at 96 hours, indicating that ~ (131) I-CD105 monoclonal antibody could accumulate in tumor tissues and reach a high level at 96 hours.
After injection of ~ (131) I marker into tail vein, the tumor site, kidney, liver and stomach began to show radioactivity concentration in the 131 I-CD105 McAb group. With the time prolonged, the tumor area became thinner in the thorax and abdomen, and gradually thickened. In the 48-hour group, the tumor area was clearly visible, the background was significantly weakened in 96 hours, and the tumor imaging was significantly enhanced.
4, the establishment and evaluation of human CD105 immunosensor detection.
On the basis of preparation of antigen (recombinant protein) and high-performance antibody, electrodeposited gold nanoparticles were used to construct biocompatible gold nanoparticles on the electrode surface, and cysteine was further modified to adsorb and modify small-sized gold nanoparticles to increase the specific surface area of working face and increase the amount of antibody immobilization. An electrochemical immunosensor for the detection of CD105 was established by immobilizing the body on the prepared electrode surface. Nano-platinum and thionine were used to improve the specificity and sensitivity of immunoassay. The electrochemical response of CD105 antibody labeled with electronic mediator is realized by thionine, and the catalytic performance of nano-platinum further improves the sensitivity of the electrochemical immunosensor. The specificity of the immunoassay system is further enhanced by detecting the specific reaction between the antibody and the antigen detected. The electrochemical characteristics of the electrode surface were investigated by cyclic voltammetry (CV), and the performance of the immunosensor was fully studied. The detection of human CD105 by CV was carried out, and good results were obtained. The electrochemical immunosensor applied to the detection of human CD105 further validates the good practicability of the electrochemical immunosensor technology, and fully proves the good detection performance and potential clinical application prospect of the recombinant protein and high-efficiency antibody. It lays the foundation for clinical application of CD105 detection technology.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R737.31

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