【摘要】：背景和目的社會人口老齡化及環(huán)境污染,致使惡性腫瘤的發(fā)病率不斷升高,成為導(dǎo)致死亡的最主要原因。在婦科腫瘤中,宮頸癌的死亡率最高,且近年來出現(xiàn)了年輕化的趨勢。尤其是在中國等亞洲國家,HPV病毒廣泛感染,導(dǎo)致宮頸癌在這些國家的發(fā)病率更高。早期宮頸癌的治療手段主要是放療,或手術(shù)聯(lián)合放療,效果較好。但是,放射線對組織的破壞作用是無選擇性的,且宮頸位于盆腔,其鄰近組織——腸道,對放射線異常敏感。因此,宮頸癌接受放療的患者常不可避免地并發(fā)放射性腸損傷,患者表現(xiàn)為腹痛、腹瀉、便血,遠(yuǎn)期甚至?xí)䦟?dǎo)致腸梗阻等嚴(yán)重情況,加重了患者心理、生理以及經(jīng)濟(jì)上的負(fù)擔(dān),也極大地加重了臨床護(hù)理的工作量。若通過降低放療劑量來避免腸道并發(fā)癥,又會導(dǎo)致宮頸癌消除不徹底,遠(yuǎn)期發(fā)生轉(zhuǎn)移和復(fù)發(fā)。因此,需要一種能增強(qiáng)宮頸癌細(xì)胞輻射敏感性的藥物,聯(lián)合放療來治療宮頸癌。以此在不影響抗腫瘤效果的情況下,可降低輻射劑量,盡可能地避免放射性腸損傷發(fā)生,進(jìn)而可減輕患者及臨床護(hù)理工作的負(fù)擔(dān)。PP2A(Protein phosphatase 2,蛋白磷酸酶)是一種參與細(xì)胞周期、DNA損傷應(yīng)答等的調(diào)節(jié)蛋白。研究證明,外源性抑制劑,如崗田酸和斑螯素,通過抑制PP2A的活性,改變腫瘤細(xì)胞的細(xì)胞周期進(jìn)程及DNA損傷應(yīng)答機(jī)制,從而增強(qiáng)腫瘤細(xì)胞對放化療的敏感性。LB-100是PP2A的一種新研發(fā)的水溶性抑制劑,動物荷瘤模型上證明其能夠有效增強(qiáng)骨肉瘤、卵巢癌等移植瘤對放化療的敏感性,且其對肝、腎等重要組織臟器無明顯毒副作用,目前處于Ⅰ期臨床研究階段。但還未見LB-100對宮頸癌作用的相關(guān)研究報(bào)道。由于宮頸癌放療常不可避免地造成放射性腸損傷,患者承受著生理和心理上的痛苦,也促使其治療和護(hù)理面臨巨大的困難,因此在研究增強(qiáng)宮頸癌放療敏感性藥物的同時(shí),應(yīng)該關(guān)注該藥物對腸道組織的作用。綜合以上因素,本研究以Ca ski(人宮頸癌細(xì)胞株)和CCD 841 Co N(人正常腸上皮細(xì)胞株)及HIEC(人正常腸上皮細(xì)胞株)為研究對象,探討了PP2A抑制劑LB-100對宮頸癌細(xì)胞株Ca ski輻射敏感性的影響,以及其對正常腸上皮細(xì)胞株CCD 841 Co N及HIEC增殖及輻射敏感性的影響。同時(shí),本研究利用小鼠放射性腸損傷模型,初步觀察了LB-100對小鼠正常腸道組織以及放射性腸損傷的腸道組織的影響,以期為宮頸癌的臨床放射治療和患者受照后的護(hù)理提供一定的指導(dǎo)。研究方法:1.LB-100藥物濃度篩選:梯度濃度的LB-100分別處理Ca ski、CCD 841 CoN和HIEC細(xì)胞,24hr后用CCK-8法檢測以上細(xì)胞的細(xì)胞活性,最終選擇對兩株人正常腸上皮細(xì)胞無抑制作用的濃度作為安全給藥濃度。2.48hr細(xì)胞活性檢測(Ca ski、CCD 841 Co N和HIEC細(xì)胞):將細(xì)胞分別分組為對照組(control)、單純LB-100組(LB)、單純輻照組(R)、LB-100聯(lián)合輻照組(LB+R),給予低濃度LB-100(2.5μM)和(或)輻照(6Gy)處理,輻照(6Gy)后48hr,以CCK-8試劑檢測細(xì)胞活性。3.平板克隆形成實(shí)驗(yàn)(Ca ski、CCD 841 Co N和HIEC細(xì)胞):細(xì)胞分組方法同2,分別給予LB-100(2.5μM)和(或)輻照(0Gy、2Gy、4Gy、6Gy)處理,24hr后去除LB-100繼續(xù)培養(yǎng),至克隆形成后進(jìn)行染色并計(jì)數(shù)。4.流式細(xì)胞術(shù)凋亡檢測(Ca ski):細(xì)胞分組方法同2,LB-100(2.5μM)和(或)輻照(6Gy)處理人宮頸癌細(xì)胞株Ca ski,3hr后給予輻照(6Gy),于輻照后24hr和48hr以流式細(xì)胞術(shù)檢測凋亡。5.流式細(xì)胞術(shù)細(xì)胞周期檢測(Ca ski):細(xì)胞分組和處理方法同4,于輻照后24hr以流式細(xì)胞術(shù)檢測細(xì)胞周期。6.免疫熒光染色(Ca ski):細(xì)胞分組為對照組(control)和單純LB-100組(LB),LB-100(2.5μM)處理48hr后,進(jìn)行免疫熒光染色(DAPI和Tublin)。7.體內(nèi)實(shí)驗(yàn):BALB/c小鼠分組為對照組(control)、單純LB-100組(LB)、單純輻照組(R)、LB-100聯(lián)合輻照組(LB+R)。給予LB-100(2.5mg/kg)或等體積生理鹽水腹腔注射,3hr后給予一次性全身輻照11Gy或0Gy。并于輻照后24hr、48hr、72hr分別給予一次LB-100(2.5mg/kg)或者等體積生理鹽水腹腔注射并觀察記錄小鼠基本狀態(tài),于輻照后84h頸部脫臼處死小鼠,獲取小鼠腸道組織,測量小腸腸度,進(jìn)行腸道組織HE和Brdu染色。結(jié)果:1.LB-100藥物濃度篩選結(jié)果顯示:當(dāng)濃度小于5μM時(shí),LB-100對以上三種細(xì)胞的細(xì)胞活性均無明顯抑制作用,當(dāng)濃度≥5μM時(shí),LB-100對以上三種細(xì)胞活性的抑制作用明顯,呈濃度依賴趨勢。2.48hr細(xì)胞活性檢測結(jié)果顯示:與control組比較,Ca ski細(xì)胞的LB組細(xì)胞活性降低,而CCD 841 Co N和HIEC的LB組細(xì)胞活性無差異,以上三種細(xì)胞的R組的細(xì)胞活性均降低。與R組比較,Ca ski的LB+R組的細(xì)胞活性降低更明顯,而CCD 841Co N和HIEC的LB+R組細(xì)胞活性與R組比較無顯著差異。3.平板克隆形成實(shí)驗(yàn)結(jié)果顯示:Ca ski和CCD 841 CoN的LB組的克隆數(shù)小于control組,而HIEC的LB組的克隆數(shù)與control組比較無差異。以上三種細(xì)胞的R組的細(xì)胞克隆數(shù)均小于control組。Ca ski的LB+R組的細(xì)胞克隆數(shù)少于R組和LB組。CD 841 CoN和HIEC的LB+R組的克隆數(shù)與R組比較無顯著差異。4.Ca ski細(xì)胞凋亡凋亡結(jié)果顯示:LB組和R組凋亡率顯著高于control組,LB+R組的凋亡率明顯高于單純LB和R組。5.Ca ski細(xì)胞周期檢測結(jié)果顯示:LB組和R組細(xì)胞周期的G0/G1期比例低于control組,但LB+R組G0/G1期比例低于LB組和R組。6.Ca ski細(xì)胞免疫熒光染色結(jié)果顯示:control組Ca ski細(xì)胞核形態(tài)正常及微管結(jié)構(gòu)完整,而LB組大量異常核出現(xiàn)且細(xì)胞核體積較control組大,微管的結(jié)構(gòu)消失。7.從小鼠的體重等基本情況、腸道組織的病理切片HE染色以及隱窩增殖染色的情況來看,LB組與control組比較無顯著差異。LB+R組與R組小鼠的體重下降,小腸長度變短,腸上皮結(jié)構(gòu)被嚴(yán)重破壞,但兩組之間比較無差異。結(jié)論:1.濃度低于5μM時(shí),LB-100對Ca ski細(xì)胞無明顯毒性作用,可選擇較低濃度LB-100(2.5μM)作為后續(xù)實(shí)驗(yàn)的給藥濃度。低濃度LB-100能增強(qiáng)宮頸癌細(xì)胞Ca ski的輻射敏感性。LB-100增強(qiáng)Ca ski細(xì)胞輻射敏感性的可能機(jī)制是誘導(dǎo)Ca ski細(xì)胞凋亡,促進(jìn)Ca ski細(xì)胞的細(xì)胞周期進(jìn)程,破壞Ca ski細(xì)胞微管結(jié)構(gòu),導(dǎo)致多倍體及異常核形成,最終導(dǎo)致Ca ski細(xì)胞發(fā)生有絲分裂性死亡。2.濃度低于5μM時(shí),LB-100對CCD 841 Co N和HIEC細(xì)胞無明顯毒性作用,視作安全濃度范圍。低濃度LB-100對正常腸上皮細(xì)胞CCD 841 Co N和HIEC的輻射敏感作用無影響。LB-100對小鼠正常腸道組織無副作用,且未加重放射性腸損傷模型小鼠的腸損傷程度,但其對損傷的腸道組織也無明顯保護(hù)作用。
[Abstract]:Background and objective social population aging and environmental pollution, resulting in the increasing incidence of malignant tumor, which has become the main cause of death. In gynecologic tumors, the mortality of cervical cancer is the highest, and in recent years, the trend of youth has emerged. Especially in Asian countries such as China, HPV virus is widely infected, causing cervical cancer in these The incidence of the country is higher. The treatment of early cervical cancer is mainly radiotherapy, or surgery combined with radiotherapy, the effect is better. However, the destruction of the tissue is not selective, and the cervix is located in the pelvic cavity, its adjacent tissue - intestinal, sensitive to radiation. For this reason, the patients receiving radiotherapy of cervical cancer are often unavoidable and Radiative intestinal injury, with abdominal pain, diarrhea, blood pressure, long term and even cause severe intestinal obstruction, aggravates the psychological, physiological and economic burden of the patient, and it also greatly aggravates the workload of clinical nursing. If the radiation dose is reduced to avoid intestinal complications, it will lead to the elimination of cervical cancer and the long term. There is a need for a drug that can enhance the radiosensitivity of cervical cancer cells and combined with radiotherapy to treat cervical cancer. In this case, the radiation dose can be reduced without affecting the antitumor effect, and the radiation intestinal damage can be avoided as far as possible, and the burden of light patients and clinical nursing work can be reduced by.PP2A (Protein pH). Osphatase 2, protein phosphatase) is a regulatory protein that participates in cell cycle, DNA damage response and so on. Studies have shown that exogenous inhibitors, such as Pantian acid and chelatosin, change the cell cycle process of tumor cells and the mechanism of DNA damage response by inhibiting the activity of PP2A, thus enhancing the sensitivity of tumor cells to radiotherapy and chemotherapy.LB-100 is PP2A A newly developed water soluble inhibitor, animal bearing tumor model proves that it can effectively enhance the sensitivity of osteosarcoma and ovarian cancer to radiotherapy and chemotherapy, and it has no obvious toxic and side effects on important organs such as liver and kidney. It is currently in phase I clinical study stage, but there is no Related Research Report on the effect of LB-100 on cervical cancer. As cervical cancer is often caused by radiation intestinal damage, patients suffer from physiological and psychological pain and are also facing great difficulties in the treatment and nursing. Therefore, we should pay attention to the role of the drug on the intestinal tissue while strengthening the radiotherapy sensitive drugs for cervical cancer. This study is based on the above factors. This study is based on Ca sk I (human cervical cancer cell line) and CCD 841 Co N (human normal intestinal epithelial cell strain) and HIEC (human normal intestinal epithelial cell strain) were studied. The effect of PP2A inhibitor LB-100 on the ski radiosensitivity of cervical cancer cell line Ca, and its effect on CCD 841 Co N, proliferation and radiosensitivity of normal intestinal epithelial cell line, were also discussed. The effect of LB-100 on normal intestinal tissue and intestinal tissue injury in mice was preliminarily studied by using the model of radiation intestinal injury in mice. In order to provide some guidance for the clinical radiation therapy of cervical cancer and the nursing care of the patients after exposure, the method of screening the concentration of 1.LB-100: the gradient concentration of LB-100 respectively Ca ski, CCD 841 CoN and HIEC cells were used to detect the cell activity of the above cells by CCK-8 method after 24hr. Finally, the concentration of the two normal intestinal epithelial cells was selected as a safe dose of.2.48hr cell activity (Ca ski, CCD 841 Co). LB), simple irradiation group (R), LB-100 combined irradiation group (LB+R), low concentration LB-100 (2.5 u M) and (or) irradiation (6Gy) treatment, and 48hr after irradiation (6Gy), and CCK-8 reagent to detect cell activity.3. flat plate clone formation experiment. After 24hr, LB-100 was removed and continued to be cultured, stained after cloning, and counted.4. flow cytometry (Ca ski). Cell grouping method was treated with 2, LB-100 (2.5 M) and / or irradiation (6Gy) to treat the Ca ski of human cervical cancer cell line, and irradiated (6Gy) after 3hr, and the apoptotic cells were detected by flow cytometry after irradiation. Cell cycle detection (Ca ski): cell grouping and treatment were 4, and cell cycle.6. immunofluorescence staining (Ca ski) was detected by flow cytometry in 24hr after irradiation. Cells were grouped into control group (control) and LB-100 group (LB), LB-100 (2.5 mu M) were treated for 48hr, and immunofluorescence staining in vivo was carried out. Group as control group (control), simple LB-100 group (LB), simple irradiation group (R), LB-100 combined irradiation group (LB+R). Give LB-100 (2.5mg/kg) or equal volume of physiological saline intraperitoneal injection, 3hr after 3hr and irradiate 11Gy or 0Gy. and irradiated 24hr. The basic state of the mice was observed and the mice were dislocated in the 84h neck after irradiation. The intestinal tissue of the mice was obtained, the intestinal intestinal degree was measured and the intestinal tissue was stained with HE and Brdu. The results showed that the concentration screening results of 1.LB-100 showed that when the concentration was less than 5 mu M, LB-100 had no obvious inhibitory effect on the cell activity of the above three cells. The inhibitory effect of LB-100 on the activity of the above three cells was obvious, and the activity of.2.48hr cells in the concentration dependent trend.2.48hr cell activity showed that the activity of LB group in Ca ski cells decreased and the activity of CCD 841 Co N and HIEC LB group was no difference compared with that of the control group. The cell activity of Ca ski LB+R group decreased more obviously, while the cell activity of LB+R group of CCD 841Co N and HIEC had no significant difference compared with the R group. The experimental results of.3. plate clone formation showed that the number of clones in Ca ski and 841 groups was less than that of the group, and the number of clones in the group was not different from that of the group. The number of cell clones was less than that of group control.Ca ski, the number of cell clones in group LB+R was less than that of R group and LB group.CD 841 CoN and HIEC LB+R group, there was no significant difference between R group and R group. The results of cell cycle detection showed that the G0/G1 phase ratio of cell cycle in group LB and R group was lower than that in group control, but the proportion of G0/G1 phase in LB+R group was lower than that of LB group and R group.6.Ca ski cell immunofluorescence staining results showed that control group was normal and the structure of microtubule was complete, while large amount of abnormal nuclei appeared and nucleus volume was larger than that of group LB+R. The structure of microtubule disappeared.7. from the basic condition of the weight of mice, the pathological section of the intestinal tissue, HE staining and the coloring of the recess, there was no significant difference between the group LB and the control group, and the weight of the mice in the.LB+R group and the R group decreased, the length of the small intestine was shorter, and the intestinal epithelium was seriously damaged, but there was no difference between the two groups. Conclusion: 1 When the concentration is less than 5 M, LB-100 has no obvious toxic effect on Ca ski cells, and the lower concentration LB-100 (2.5 u M) is selected as the dosage of the following experiment. The low concentration LB-100 can enhance the radiosensitivity of Ca ski in cervical cancer cells, and the possible mechanism of enhancing the radiosensitivity of Ca ski cells is to induce apoptosis and promote the cell apoptosis. The cell cycle process disrupts the microtubule structure of Ca ski cells and causes polyploidy and abnormal nucleus formation, which eventually leads to the mitotic death of Ca ski cells and the.2. concentration is lower than 5 u M, and LB-100 has no obvious toxic effect on CCD 841 Co N and HIEC cells, which is considered as a safe concentration range. Low concentration LB-100 is 841 of normal intestinal epithelial cells. The radiation sensitivity of C did not affect the side effects of.LB-100 on normal intestinal tissue in mice, and did not aggravate the degree of intestinal damage in mice with radiation intestinal damage, but it had no significant protective effect on the injured intestinal tissue.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.33

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