Radioresistence And Chemoresistence Of Glioma Stem Cell In Vitro

In the whole body malignant tumor, The5-years’ mortality of malignant glioma are after pancreatic cancer and lung cancer, the5-year survival rate are less than5%. Brain glioma account for more than half of intracranial tumors, the incidence of cerebral glioma is3-10/100,000, There is1-3%of whole malignant tumors. Low grade glioma are near a benign pathologically and less invasive. even so, there are still nearly half of the patients whose survival time was less than5years, the malignant gliomas with low grade will develop to higher grade of malignant gliomas, almost all patients died of tumor recurrence and/or local invasion finally,Surgical excision of glioma is the most basic and most direct way of treatment, treatment effect is clear, the main purpose of surggery is to reduce morbidity and mortality and improve patients’quality of life and survival time to greatest degree But as a result from infused grow,it is difficult to distinguish the clear boundary by naked eye in the operation procedure, it is difficult to resect completely, it is so often to need radiotherapy and chemotherapy after surgery. Another part of patients because of smaller lesions, or lesions located in functional area, or because the older, poor general condition of body, they were refused or unfavorable surgical operation therapy and radiotherapy or chemotherapy was only treatment choice. Irradiation therapy is mainly making biological cells ionized by using properties of irradiation penetrating.commonly irradiation were used such as x-rays and gamma knife, X-ray and gamma knife passed the ionizing irradiation energy into the organization and interacted with the atomic of living organisms,. Bioactive tissues produce a series of complicated physical, chemical reactionl after radiation energy ionized, and biological organization would lead to radioactive damage. Radiation therapy including ordinary radiotherapy, irradiation closely to organization and precise radiotherapy, radiotherapy precisely, including three-dimensional conformal and intensity modulated radiotherapy and stereotactic radiotherapy (i.e., X knife, gamma knife). Either radiotherapy methods lead to tumor tissues and cells ionizing damage by using x-rays, gamma rays to achieve therapeutic effectssurgery and chemical therapy, radiation therapy make up the system for the treatment of glioma, the chemical treatment can kill the small lesions after surgery or radiotherapy, and delay the recurrence of gliomas; Sometimes chemical treatment can make patients who can’t accept surgery originally or are not suitable for surgery can undergo surgery. But chemical therapy also has the side effects, systemic drug toxicity is larger, at the same time chemical therapy may cause inhibition to the patient’s immune system. Chemical drug is divided into two categories, respectively, the cell cycle specific or cell cycle non-specific drugs. When choosing a combination chemotherapy often consider two classes of drugs used in combination.Though the comprehensive treatment of gliomas includ surgery, radiotherapy and chemotherapy and gene therapy conducting clinical research currently, immune therapy, etc. but the curative effect of glioma get no significant progress in more than ten years, finally patient almost died of local invasion or local recurrence. Especially poor outcome of malignant glioma, even undergoing surgery and radiation and chemotherapy, the median survival time are only14.6months. At present neurosurgery scholars generally believe that the key problem in treatment of glioma is that existing treatment cannot effectively inhibit or delay the tumor recurrence.Recent research think that the tumor is composed of a of stem cells that have capacity of unlimited proliferation and self-renewal and cell groups that are differ-entiated, inhomogeneity cells, the small population of stem cells in tumor is known as tumor stem cell, TSC. The principal part of the Tumor is composed of cell lost self-renewal ability,differentiated characteristics. The most important features of cancer stem cell hypothesis is that tumor were generated by stem cells and stem cells lead to recurrence after treatment. Theory of cancer stem cell is is of great signific-ance for understanding the essence of the tumor and the exploiting of new cancer treatment. Including surgery, radiotherapy and chemotherapy, traditional treatment served the whole tumor as a treatment target, and there is no specific for cancer stem cells. The final result of treatment most likely remain tumor stem cells with tumori-genicity and stronger resistance to therapy, lead to tumor relapse easily, it is difficult to cureGlioma stem cells are gradually on the basis of this theory, the earliest known as neural stem-like cell., The significance of the theory of glioma stem cells is that if we can specific identify of glioma stem cells, and develop the specificity for the targeted treatment of glioma stem cells, we may thoroughly conquest of glioma.According to the theory of cancer stem cells and glioma stem cells, there are glioma stem cells in brain tumor tissue and glioma cell line cultured in vitro, and because the glioma stem cells has certain resistance effect for the treatment of the current method and result in glioma stem cells survived after treatment, lead to glioma recurrence after treatment. This tissue is designed on such way of thinking.At first in the first section, two specimen from malignant glioma tissues freshly and U251cells were inoculated in serum containing medium, after cells growth well, the cell were inoculated to serum-free medium (DMEM/F12medium, containing B27,20ul/ml, bFGF20ng/ml, EGF20ng/ml, LIF10ng/ml),then collect tumor spheroid suspending growth, the suspending tumor spheroid were cultivated in serum-free medium, after amplified, to test stem cell surface marker CD133and ABCG2expression by immunofluorescence cytochemistry method; to detect expression of CD133and ABCG2By flow cytometry in suspending tumor spheriod; to sort out the CD133positive cell in tumor spheroid by magnetic bead labelled with CD133antibody;, single cell clone forming method and tumorigenic cells transplantation into nude mice were done to detect its stem cell characteristics. In results, we found that there were suspending floating sphere cultivated in serum-free medium in the fresh glioma tissue and U251cell line; the sphere can be found that have obvious expression of CD133and ABCG2detected by immunofluorescence cytochemistry; But to test expression of CD133and ABCG2by flow cytometry.the CD133+cells U251cells is only3.17%, while the ABCG2+cells was only1.17%in U251sphere cells; And in fresh glioma tissue cells, the positive expression rate of17.8,9.68%,11.25%and14.2%, respectively. After magnetic bead sorting labeled by CD133antibody, CD133positive expression were88.2%,91.5%and87.4%respectively. the cell clone forming test was carried out respectively, the results show that CD133+cells clone forming rate were78.592.4%, and CD133-cells were only1.5--4.7%. Transplanted by CD133+cells density of102-103/ml, CD133-1-5×105cells/ml to1ml into nude mice abdomen, tumor formed by CD133+cells reached to80%(4/5). In the first section, we found that:in glioma tissues and U251glioma cell line, there are a small number of glioma stem cell expressed surface marker CD133and ABCG2,which can maintain self-renew and the ability to infinite proliferation in vitro; glioma stem cells can be purified by magnetic bead separation labelled by CD133antibody; cell clone forming rate and tumor forming rate nude of CD33+cells is higher than CD133-cells. Therefore, we can think that the small number of cell expressed surface marker are glioma stem cells.In the second section, identified glioma stem cells are treated by X-ray irradiation, cell activity of different time after irradiation were determined by MTT colorimetry; Expression of ABCG2and CD133were detected in72hours after irradiation, cell nucleus were stained by Hochest3325to detect of apoptotic; apoptosis were detected by the Annexin V-FITC/PI apoptosis kit after irradiation; At last we will pair cells treated72hours after6Gy irradiation with cells without X-ray irradiation for temozolomide(TMZ) treatment in half inhibition concentration for48hours. the cell activity were determined by MTT colorimetry and cell apoptotic rate were detected by Annexin V-FITC/PI apoptosis kit, thue we will estimate combination effect of radiotherapy and chemotherapy for glioma stem cells. Results showed that:1the activity of U251sphere cells and U251cells decreased significantly (P<0.01)with the increase of radiation dose and treatment time extended, Under the same dose of different time activity of cell significantly decreased (P<0.01), but in the3Gy radiation dose, activity of U251cells between48h and72h difference was not significant (P=0.057);2At72h after irradiation treatment, expression of CD133and ABCG2detected by Flow cytometer, results show that,72h after the3Gy,6Gy,9Gy X-ray irradiation, the expression of CD133from3.17%to4.74%,8.35%,13.24%and expression of ABCG2from1.17%to1.48%,3.51%and8.76%, respectively.3, each phase of cell apoptosis by hochest staining were showed at72h after irradiation;4cells apoptotic rate of the CD133+CD133-cells U251sphere cells and U251cells were tested by Annexin V-FITC/PI apoptosis kit respectively, Apoptotic rates of CD133+cells at72h after irradiation for3Gy,6Gy,9Gy owere2.64+0.82%,4.53±1.19%and6.07±1.52%; CD33- cells were9.73±0.92%,17.94±1.33%and25.19±1.56%; It was found that,at72hours after irradiation apoptotic rate of CD133+cells, between3Gy and6Gy irradiation dose and6Gy and9Gy dose were no difference (P>0.05) After statistical analysis,3Gy and9Gy, apoptosis rate have significant difference (P<0.01); apoptotic rate of CD133+cells, CD133-cells, U251cells and U251sphere cells in the same X-ray doses have significant difference (P<0.01); apoptotic rate of U251sphere cells,CD133-cells and U251cells in the same X-ray dose have significant difference; U251cells and CD133-cells in the same irradiation dose,apoptosis rate was no statistical difference (P>0.05).5paired cells treated72hours after6Gy irradiation with cells without X-ray irradiation for temozolomide(TMZ) treatment in half inhibition concentration for48hours. Activity of cell were measured by MTT colorimetry and apoptotic rate were detected by Annexin V-FITC/PI apoptosis kit at before TMZ treatment and48h after TMZ treatment respectively. Results show that the OD value of the sphere, the sphere/IR, CD133+,CD133+/IR and CD133-, CD133-/IR cells measured by MTT was1.241±0.11,0.785±0.055,1.257±0.08,1.147±0.084,1.241±0.103,0.568±0.046before TMZ treatment; it was0.569±0.032、0.424±0.036、1.142±0.071、0.907±0.042、0.632±0.063、0.234±0.040; Apoptotic rate was0.53±0.12%,12.63±2.0%,0.40±0.09%,4.58±0.52%,0.60±0.4%,17.91±2.0%before the TMZ treatment respectively:it was18,75±2.7%、51.99±5.2%、2.62±0.7%、17.09±1.9%、21.98±3.4%.42.33±3.5%at48h after TMZ treatment. By paired T test and single factor analysis of variance,it showed that the change of for CD133+cells between befoe and after TMZ treatment were no statistical difference; The OD value of rest group in different cells, were significant difference before and after drug treatment (P<0.01); Compared with CD133+cells, changes of OD value of rest cells group before and after TMZ treatment measured by MTT colorimetry were significant difference (P<0.01); But cells group after radiation treatment, the change of OD value was no statistical difference; Compared with CD133+cells, apoptotic rate of rest groups before and after drug treatment were a significant difference (P<0.01). In the second section of the research, we found that:in different dose of X-ray irradiation, the activity of cells decreased with the increase of radiation dose and extension of time, apoptotic rate increased with the increase of radiation dose, and gradually increase with the extension of time; radiation dose increasing, the expression of stem cell markers in spheroid were increase; In72h after radiation treatment, apoptotic rate of CD133+cells treated in3Gy and6Gy,6Gy and9Gy was no significant difference. The result prompted that gliioma stem cells has a certain resistance to X-ray irradiation.therefore, the apoptosis rate was not increased with the increase of irradiation dose after radiation treatment. We paired CD133+cells, CD133-cells dnd tumor sphere cells after irradiation with cells without irradiation treatment, activity of cells and apoptotic rate were measured before and after TMZ treatment respectively. We found that glioma stem cells marked by CD133were resistance toTMZ treatment. Compared with the CD133+cells after radiation, apoptotic rate and activity of cells by measured by MTT before and after’TMZ treat have significant differenceTo sum up, the experimental results show that there is a small population of the glioma stem cells in brain glioma tissue and U251glioma cell line cultured in vitro for a long time, expressing stem cell marker CD133and ABCG2. Glioma stem cell can be proliferation repeatedly in vitro. Transplantation in null mice will form tumor strongly. Glioma stem cell show a certain resistance to sole chemotherapy or sole radiotherapy. But combination of chemotherapy and radiotherapy will decline resistance of glioma stem cell.