| Background and ObjectiveCeretral glioma is the No.1 brain malignancy worldwide. Though the operative treatment of the glioma has improved for the development of neuroimageology and microsurgery techniques, and gene therapy would be a promise way with lucubrated of molecular biology advancements. However, there are no great breakthroughs in the glioma therapy by now. The integrated therapy of operation, radiotherapy and chemotherapy is the primary method to treat the gliomas. Thus, how to improve the effect and quality of life and prolong the survival become the tough problems that neurosurgens need to solve.Chemotherapy is an important component in glioma therapy, which can eliminate the residual glioma cells and delay the glioma recrudescence. The selection of chemotherapy drugs and treatment programs has great impact on therapeutic effects. Nowadays, the individualized tumor treatment based on chemosensitivity assay in vitro has been the leading tendency. Select more effective drugs by chemosensitivity assay before the chemotherapy can enhance the therapeutic effects, avoid the side-effects and toxicity response and save the expenses.It is well-known that there were different kinds of cells in glioma tissue, such as astrocytes, nerve cells, endothelial cells, blood cells and glioma cells. Among them, the glioma cells and astrocyes were the majority. Because different cells showed different sensitivity to the same chemotherapy drugs, it may influence the interpretation of results. Further understanding about the chemotherapy drugs' effects on proliferations of the glioma cells and astrocytes was an important reference to select the concentration of drugs and the timing of detection, which consequently enhanced the creditability of the results and the efficacy of clinical treatment.In our study, firstly, U251 cells and human fetal astrocytes were selected as research objects and six drugs that were frequently used in clinical chemotherapy were tested, including MeCCNU, DDP, VM26, VP16, VCR, CTX. The growth curves and cells cycle and inhibition rate with cultured time were detected under different concentrations of 6 drugs above. The suppression of cell proliferation was analyzed. The results were the reference to choose the drug concentrations and the timing of examination in the study. Secondly, the fresh glioma samples were collected and the glioma cells were cultured in vitro. The morphologic characters and the changes of cell growth of the glioma cells from different pathological grades were observed. Immunocytochemistry assay was used to identify the antigens of glioma cells. Chromosome G-banding technique was uesd to show chromosome karyotype. The differences of the glioma cells biological characters among each pathological grade were investigated. Brain tumor stem cells were also cultured in this study. Finally, we chose the patients who were operated. Whose glioma cells were cultured in vitro. We selected the sensitive drugs from MeCCNU, DDP, VM26, VP16, VCR and CTX used in chemotherapy based on the results of chemosensitivity assay.The results were as follows:1. U251 cells growed rapidly and human fetal astrocytes did slowly. The cells group double time was 3.8d and 5.86d respectively. The results of cell cycle assay showed that the percentage of G0/G1 phase, S and G2/M phage of U251 and astrocytes was 39%, 28.78%, 32.22% and 80.21%, 9.68%, 10.11%.2. The cells growth curves and double time of U251 and astrocytes cultured under MeCCNU and CTX were similar to the control groups.3. The proliferation of U251 and astrocytes was restrained slightly following the action of MeCCNU与CTX. The inhibitive rate showed an increasing tendency with the concentration and the cultured time, most of that was lower than 20%.4. U251 cells were cultured with VM26. The double time of 0.1 X PPC group was 4.91d. It was prolonged 1.11d compared to the control group. The astrocytes were cultured with VM26.The double time of the 0.11×PPC and 0.5×PPC group was 9.4d and 11.46d, postponded 3.54d and 5.6d compared to the control group respectively. The number of cells was obviously reduced in the other groups, especially in 5×PPC and 10×PPC group.5. U251 cells were cultured with VM26. The inhibitive rate of each concentration group was 24.16%, 21.31%, 23.69%, 28.15%, 31.29%和16.5%, 27.44%, 31.74%, 29.55%, 25.58% in cultured l~2d. The proliferation was limited remarkably, but there was not different among each group. In cultured 3~7d, the extent of cell growth inhibition was developed prominently with the increasing concentration of VM26 and the cultured time, especially in 5×PPC and 10×PPC group. The inhibitive rate was 79.71% and 99.56% in 5×PPC and 10×PPC group in cultured 4d. The effect of VM26 was changed gently when they cultured after 4d. The astrocytes were cultured with VM26. The inhibitive rate of each group was 3.29%, 7.09%, 3.29%, 9.32%, 66.05% at first day. The rates of the groups were inceased with the cultured time, but it showed dissimilarity between the groups. In 0.1×PPC group, the rate was kept on a low level in cultured 1～7d, that was less than 15%. In 0.5×PPC and 1×PPC group the rate was climbed slightly in 1～4d but rised sharply after 4d. The rate of 0.5×PPC and 1×PPC group was near 50% in cultued 6d and 5d respectively. In 5×PPC and 10×PPC group the rate was ascended sharply with time. The rate of the groups was more than 90% after cultured 4d and 3d.6. U251 cells were cultured with VP16. The cells growth was not influenced and the double time was 3.61d in 0.1×PPC group. The double time of the 0.5×PPC and 1×PPC group was 9.07d and 8.06d, postponded 4.87d and 3.87d compared to the control group respectively. The astrocytes were cultured with VP16. The double time of 0.1×PPC, 0.5×PPC and 1×PPC group was 7.39d, 7.79d, 9.02d, delayed 1.53d, 1.93d, 3.16d compared to the control group. The number of U251 cells and astrocytes was reduced in 5×PPC and 10×PPC group.7. U251 cells were cultured with VP16.The inhibitive rate of each concentration group was 19.13%, 15.89%, 31.67%, 23.97%, 29.1% at first day. The cells growth was limited with concentration, but there was little difference among each group. The gap was less than 15.78%. In cultured 2~7d, the extent of cell growth inhibition was developed prominently with cultured time, and the maximal gap was 36.44%, 40.96%, 57.57%, 75.56%, 81.07% respectively. There was trifle difference between 0.5×PPC and 1×PPC , 5×PPC and 10×PPC group. The astrocytes were cultured with VP16. The inhibitive rate of each group was 1.28%, 9.77%, 24.51%, 15.8%, 19.6%. There was little difference among 1×PPC, 5×PPC, 10×PPC group. Following the cultured time, the inhibitive rate was increased slowly in 0.1×PPC group and it was 14.73% at cultured 7d. In 0.5×PPC and 1×PPC group, the rate was accelerated, which reached 35.97% and 35.17% at cultured 6d. There was similar between 0.5×PPC and 1×PPC group. The rate was risen sharply in 5×PPC and 10×PPC group. The maximal rate was 68.59% and 97.99%.8. The effect of VCR was displayed to bring the cells into death. The number of U251 cells and astrocytes was decreased gradually after cultured 2d. There was not markable difference among the groups. The number of U251 cells groups was decreased 9.38%, 9.04%, 2.05%, 27.56%, 16.44% at seventh day and that of astrocytes was reduced to 90.53%, 75.31%, 66.11%, 65.21%, 56.06% at sixth day in each groups respectively.9. U251 cells were cultured with VCR. The cells growth was limited and the linear tendency was showed with the cultured time. The inhibitive rate of each groups was 2.78%, 13.14%, 12.09%, 5.16%, 7.53% at first day and that was 51.87%, 57.49%, 55.51%, 48.75%, 47.74% at fourth day and the one was 68%, 74.46%, 68.73%, 75.04%, 71.94% at seventh day respectively. The astrocytes were cultured with VCR. The inhibitive rate of each groups was 7.98%, 10.44%, 20.27%, 18.48%, 20.71% at first day and the one was 49.93%, 59.46%, 68.32%, 68.05%, 73.29% at sixth day. The effect of inhibition was increased with the cultured time. There were not diffenence among each groups of U251 cells and astrocytes.10. U251 cells were cultured with DDR The cells growth of 0.1×PPC group was not different to the control group and the double time was 4.43d. The number of U251 cells was decreased in the other groups after cultured 2d. The extent of cells reducer was related to the concentration. The cells in 5×PPC and 10×PPC group was near vanished at sixth day. The astrocytes were cultured with DDP. The cells number was developed slowly in 0.1×PPC, 0.5×PPC and 1×PPC group and the double time was 6.24d, 7.38d, 8.59d respectively. At seven day, the cells number was 16% and 19% to the beginning in 5×PPC and 10×PPC group.11. U251 cells were cultured with DDP. The inhibitive rate of each groups was 7.68%, 9.28%, 5.12%, 27.36%, 17.28% at first day. The rate of 5×PPC and 10×PPC group was more than the one of the others. The rate was 20.4%, 71.27%, 84.81%, 94.14%, 93.78% and 19.79%, 84.96%, 95.34%, 99.89%, 99.94% at fourth day and seventh day. The cells growth was obviously limited in the groups but 0.1×PPC group. There were less different among the groups after cultured 4d. The astrocytes were cultured with DDP. The inhibitive rate of each groups was 1.76%, 1.96%, 9.61%, 50.98%, 58.82% at first day. The rate of 5×PPC and 10×PPC group was more than the one of the others. The inhibition was increased continually with the cultured time in each groups. The rate range was 30% to 40% in 0.1×PPC group, and the one was 50% to 70% in 0.5× PPC and 1×PPC group, and that was 90% to 100% in 5×PPC and 10×PPC group.12. After U251 cells cultured with the drugs for four days, the cell cycle assay showed that there was not influence with MeCCNU and CTX, and the percentage of G0/G1 phase was decreased, and that of G2/M phase was increased with VCR and VM26. With DDP and VP16, the percentage of G0/G1 phase was decreased, and that of G2/M phase and S phase was increased. After astrocytes cultured with the drugs for four days, the cell cycle assay showed that there was no chang with MeCCNU and CTX, and the percentage of G0/G1 phase raised, and that of G2/M phase decreased with VM26. With DDP and VCR, the percentage of G0/G1 phase decreased, but that of G2/M phase increased with VCR and that of S phase did with DDP. The percentage of S phase was decreased and the one of G2/M phase was developed with VP16 .13. The shape of most of glioma cells in primary culture presented rotundity, shuttle and stelliform. At early phase, rotundity cells were the majority, while with the cultured time prolonged the number of shuttle cells increased.14. The proliferation of astrocytes was correlated with pathological grade. The cells growed slower at lower grade and faster at higher grade. The double time of astrocytes was ranged from 3 to 5 days.15. There were remarkable differences in cell cycle among III—IV grade astrocytes. The percentage of G0/G1 phase may ranged from 50.33% to 94.7% and that of S phase from 2.9% to 14.18% and that of G2/M phase from 2.27% to 35.48%.16. The results of chromosome karyotype showed that the abnormality of chromosome number and chromosome structure could be observed in glioma cells, and the abnormality of sex chromosome amount and the multiplication of 7th chromosome and the loss of 17th chromosome were more frequent.17. 27 glioma patients were enrolled in this study to estimate the sensitivity to the chemotherapy drugs. 3 cases showed no sensitivity to all drugs and 5 cases were sensitive to 1 drug and 4 cases were sensitive to 2 drugs. 7 cases were sensitive to three or four drugs. Only 1 case was sensitive to all drugs. 16 cases used the recommended drugs by sensitivity assay in clinical chemotherapy.18. The results of the effects on glioma cells treated by drugs showed that the cells' sensitivity to CTX and MeCCNU were the lowest among all drugs. Only 1 case demonstrated low level sensitivity to CTX and MeCCNU. The glioma cells demonstrated the highest sensitivity to VCR and 22 cases were sensitive to it. 17 cases were sensitive to VM26 and 15 cases were sensitive to VP16 and 12 cases were sensitive to DDP.19. To investigate the sensitivity of different pathological grade glioma cells to the drugs, the results showed that 9 cases I—II grade astrocytoma cells were not sensitive to CTX and MeCCNU. The number of cases that was sensitive to DDP, VM26, VP16, VCR was 3, 4, 2, 7, in which there were 1, 1, 0, 2 cases presented morderate level sensitive to DDP, VM26, VP16, VCR and there were 2, 3, 2, 5 cases revealed low level sensitive to DDP, VM26, VP16, VCR. In 8 cases II-III grade glioma cells, only 1 case was sensitive to CTX and MeCCNU. The number of cases that was sensitive to DDP, VM26, VP16, VCR was 6, 7, 6, 7, in which there were 1, 2, 2, 2 cases showed high level sensitive to DDP, VM26, VP16, VCR and there were 2, 3, 1, 3 cases presented moderate level sensitive to DDP, VM26, VP16, VCR and there were 3, 2, 3, 2 cases revealed low level sensitive to DDP, VM26, VP16, VCR. In 10 cases III-IV grade glioma cells, no one was sensitive to CTX and MeCCNU. The number of cases that was sensitive to DDP, VM26, VP16, VCR was 6, 6, 5, 8, in which there were 1,2, 1, 1 cases showed high level sensitivity to DDP, VM26, VP16, VCR and there were 1,2, 1,2 cases presented moderate level sensitivity to DDP, VM26, VP16, VCR and there were 4, 2, 3, 5 cases showed low level sensitivity to DDP, VM26, VP16,VCR.Conclusions1. Because CTX and MeCCNU need to be metabolized into potent components in liver which could inhibit the cells growth, the effect would not be presented in chemosensitivity assay. They were eliminated in the study. DDP, VM26, VP16, VCR showed the inhibitive action to glioma cells and astrocytes. To compare the effects between U251 cells and astrocytes, the optimal concentrations and timings were as follows:2. The astrocytoma cells growth was correlated with the pathological grade in primary culture. The higher the malignance was, the faster the cells grew. The abnormal chromosome karyotype was observed in glioma cells. The glioma stem cells were cultured from III ~ IV grade astrocytoma.3. About 60% of glioma patients could be obtained the sensitive drug filtrated by chemosensitivity assay in vitro. In a word, the glioma cells showed lower sensitivity to the antitumor drugs, in which most of them presented moderate sensitivity and showed the sensitivity to one or two drugs. VCR and VM26 showed better sensitivity than other drugs to the glioma cells.
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