In Vitro Chemosensitivity Testing Of Bgscs On Chemotherapy Agents

Background and Objective:Ceretral glioma is the No.1 brain malignancy worldwide. Nearly, though the techniques of diagnose and operative treatment of the glioma has improved, gene therapy and microsurgery techniques has got a lot of progress, the therapy for glioma has no great breakthroughs, and the prognosis of the patients are still not satisfied. Most patients(about 70%) who is ill with the malignant glioma usually dead after the diagnosis. Nowadays, the primary therapy techniques for glioma are integrated therapy of operation, radiotherapy and chemotherapy. Thus, how to improve the effect and quality of life and prolong the survival become the tough problems that neurosurgeon to solve.As the development of tumor research, the researchers find that: there are small part of cells in the tumor which termed "tumor stem cells" have the ability of self-updating, the abnormal proliferation in the mainly development of tumor. Most of tile tumor cells lose the ability to proliferate and self renew and they differentiate into tumor cells that become the phenotypic signature of the tumor. The tumor stem cells theory had been verified since the tumor cells were isolated from Leucocythemia and breast cancer.Brain tumor stem cells theory suggests that cells similar to NSC existed in brain tumor. They can form brain tumor sphere(BTS) in vitro SFM and have abilities of self-renewal, abnormal proliferation and differentiation potentiality. They can differentiate into neural and galotinous similar to cell proportion of primarily tumor in certain environment. They are a little fraction expressed CD133 in brain tumor and termed brain tumor stem cell(BGSC). If 100 of these cells were transplanted into NOD-SCID mice brain, we'll find a new tumor which similar to the formal one formed there after 3-6 months later. So, the finally way to cure brain tumor is vanish all the BGSC.Now, the best way to treat brain tumor is: under that precondition of safe neurology, mostly wide-ranging ablating tumor tissue, then combined with effective radiotherapy and chemotherapy. The selection of chemotherapy drugs and treatment programs had great impact on therapeutic effects, improve the quality of patients' life, and prolong the survival. Nowadays, the individualized tumor treatment based on chemosensitivity assay in vitro has been the leading tendency. Select more effective drugs by chemosensitvity assay before the chemotherapy can enhance the therapeutic effects, avoid the side-effects and toxicity response and save the expenses.Design of experiment is carried out based on above theory and clinical real apply. To isolate, cultivate and identified brain tumor stem cells(BGSC) in glioma, then, we select four kind of chemotherapy drugs: TMZ(temozolomide) RPM(rapamycin) ATO(arsenious acid) and DDP(cisplatin) which used in the in vitro chemosensitivity testing for glioma, and observe the change of BGSC's inhibitive rate, and analysis the extend of the drugs sensitivity, to certified the significance of the in vitro chemosensitivity testing for glioma, and provide the evidence for clinical apply. TMZ is a kind of new oral chemotherapy drug which has quickly-absorb, whole-absorb character and can go through the blood-brain barrier; RPM is a kind of new effect, low toxic, no renal toxic immunosuppressant drug. Nearly, it was found has the effect for ant-cancer. ATO is the main components of arsenic which is a kind of traditional Chinese medicine of our country. DDP is a kind of clinic first-line chemotherapy drug.Methods:Under sterile conditions for surgical resection of the pathological examination confirmed II～IV-class glioma specimens, PBS fully washed after with 0.125 % of trypsin to digest at 37℃30min,thenYansui into single cell suspension to 2×10~5 / ml concentration in the preparation of good inoculation serum-free medium which contend EGF and bFGF Conduct training. We observe the BGSC's growth by phase contrast microscopes, and test the expression of Nestin GFAP by IHC, and test the expression of CD133 by immunofluorescence. After conformed BGSC, we cultured BGSC with different dose[ppc plasma pleak concentration(μg/ml)](10ppc 5ppc 1ppc 0.5ppc 0.1ppc 0ppc) of TMZ RPM ATO and DDP separately, then test the inhibitive rate after 24h 48h and 72h. The change of morphology can be observed by phase contrast microscopes, test the inhibitive rate by MTT, and the apoptosis can be tested by Caspase-3 SP-kit, analyzing statistical significance with analysis of variance(One Way ANOVA), and making multiple comparison with the least significant difference(LSD).Results:There are BGSC in glioma tissue, which can be isolated and cultured in vitro; the single cell suspension culture bottle in the train after the formation of large and small 24～48 h, the refractive index of strong, suspended the growth of tumor stem cells ball; 4～5 d after the passage of the visible tumor-forming stem cells, the number gradually increased, the volume increases , Shape the rules, tumor cells and stem cells-the same as the original form.Immunofluorescence staining CD133 and IHC staining Nestin GFAP positive staining; BGSC cell cycle analysis with stem cell characteristics of a typical cycle, most cells in the G0/G1 phase, and only a few cells into the S phase.The BGSC has the character of differentiation induction, grow from around, and cell morphology was clostridium article, similar to human glioma cell-line.Different dose of TMZ can inhibit BGSC, when the dose is lppc, the inhibitive rate is (64.3±1.7) % and (24.1±2.4) % partly at 72h and 48h, and the two phases' difference has statistical significance(P<0.01); Different dose of RPM can inhibit BGSC, when the dose is lppc, the inhibitive rate is (37.8±0.4) % and (29.1±0.6) % partly at 72h and 48h, and the two phases' difference has statistical significance (P<0.01); Different dose of ATO can inhibit BGSC, when the dose is 1ppc, the inhibitive rate is (62.6±3.0) % and (40.5±2.2) % partly at 72h and 48h, and the two phases' difference has statistical significance(P<0.01); Different dose of DDP can inhibit BGSC, when the dose is 1ppc, the inhibitive rate is (63.5±1.3) % and (32.7±1.7) % partly at 72h and 48h, and the two phases' difference has statistical significance(P<0.01); As the increase of dose and prolong time, the inhibitive rate gradually higher, and show Time-Dose Effect. When the dose is 1ppc, the ATO's inhibitive rate is much higher than TMZ and RPM's, the difference has statistical significance after 24h; When the dose is 1ppc, the ATO and DDP's inhibitive rate is much higher than TMZ and RPM's, the difference has statistical significance after 48h; When the dose is 1ppc, the ATO DDP and TMZ's inhibitive rate is much higher than RPM's, the difference has statistical significance after 72h; so, we can find that ATO and DDP are more effective than TMZ and RPM in early time, but TMZ can catch up the two kinds of drugs in later time, and much more effective than RPM. Because of TMZ's good points, it has better long-term effect than others. TMZ RPM ATO and DDP's inhibitive rate is different for every case of all the 13 cases.After be treated, the BGSC's modality changed, a lot of cells scatter from the BTS, and weak in refractive index.Conclusion:1. BGSC can be isolated from glioma tissue, and BGSC express stem cell marker such as CD133 Nestin ,and they can cultured in vitro, with self-renewal and proliferation of cloning.2. TMZ RPM ATO and DDP affect BGSC in Time-Dose Effect: with the increase of dose and prolong time the inhibitive rate gradually higher. ATO and DDP are more effective than TMZ and RPM in early time, but TMZ can catch up the two kinds of drugs in later time, and much more effective than RPM.3. TMZ RPM ATO and DDP, every kind of drugs has different inhibitive rate for different original BTSC, even in similar time and similar dose.4. TMZ RPM ATO and DDP can inhibit the growth of BGSC, and induce apoptosis. There is the activation of Caspase-3 zymogen protein at the whole process of cells apoptosis.