A Preliminary Study About Simulated Microgravity And Dl-THP On Glioma Cell Proliferation, Apoptosis Effect And Mechanism

Malignant glioma is one of the most harmfulintracranial tumors to human health. Because of its infiltrative growth, no obvious boundary with brain tissue, and the surgery is difficult to cut the whole.But recurrence rate is qiute high while the survival rate is low.As we all known,the common antineoplastic drugs used today are quite harmful to the body as well as strong side effects.What’s more,the tumor cell is easily to have a resistance,which has a bad effects for therapy [1]. Therefore, methods and drug therapy of malignant gliomas has become a hot issue in the treatment of malignant glioma [2]. The study confirmed that the way drug treatment of malignant gliomas is mainly depend on inducing cell apoptosis.As a consequence, to investigate the effect of treatment and drugs on apoptosis of glioma cells has important significance.Because of the important role of traditional Chinese medicine which has a lifting effect on cancer prevention and treatment.Thus, we intend to search the high-frequency drugs from the multitude of ancient books of traditional Chinese medicine to find some elevating role in the treatment of malignant gliomas,and to observe its antitumor effect.In this way, we can make contribute to both simulated microgravity and traditional Chinese medicineWith the development of space life science, researchers found that microgravity orsimulated microgravity can slow cell growth, influence the cycle,and promote its apoptosis.For malignant glioma cells have thecharacteristics of cell proliferation raipedly, cell cycle uncontrolled and anti apoptotic. Therefore, we adopt the method of simulated microgravity intervention in malignant glioma cell U251 MG, observe its effect on cell proliferation, cell cycle and cell apoptosis.Part 1 High-frequency medicines analysison malignant glioma treatment which from ancient Chinese medicine recordsObjective: we have to screen drugs in ancient medical books,whichboth have the lifting function andanti-malignant glioma effect.Methods:Using text mining method, collecting ancient medical books which have the treatment of brain tumors, and extracting cases; the use of Microsoft Access software to establish the database of ancient medical records brain tumor disease, mining and analysis of high frequency drugs on the treatment of malignant gliomas statistical method using SAS 9.1 statistical software,and filter out the high-frequency drugs in the treatment of brain malignant glioma.Results: Among the 343 ancient books, records a total of 143 traditional Chinese medicines, high drug concentration in the first 32 flavors, of which the cumulative frequency up to 77.53%. There are 11 flavors traditional Chinese medicine with lifting function,accounting for the entire high-frequency drug group 34.37%. The highest frequency one is Yanhusuo corydalis.Conclusion: The Yabhusuo Corydalis with lifting function is selected as the highest frequency of Chinese medicine treatment of brain tumor.Part 2 The study of dl-THP anti-malignant glioma effect and its ralated mechanism The first experiment:the effect and molecular mechanism of dl-THP in apoptosis of human malignant glioma U251 MG cells.Objective: To observe the effects of different concentrations of dl-THP on U251 MG cells apoptosis and expression of P53 and itsdownstream factor p21.Methods: Different concentrations of dl-THP were intervented on U251 MG cells, CCK-8method was used to detect the proliferation capacity of U251 MG, flow cytometry and TUNEL method were used to detect apoptosis; protein immunoblotting method were used to detect apoptosis related protein p53 and p21.Meanwhile, using the p53 inhibitors PFTalpha combine with dl-THP treat on U251 MG cells to observe the proliferation and apoptosis of U251 MG cells.Results:(1)After the intervention of dl-THP U251 MG cells at different concentration, the proliferation of U251 MG cells were inhibited significantly(P<0.05), and in a concentration dependent manner;(2)the apoptosis rate of U251 MG cells increased with the concentration of intervention(P<0.05);(3) The expression of p21 and P53 of U251 MG cells up-regulation(P<0.01), after the combined treatment of dl-THP and PFT- alpha, apoptosis and proliferation of U251 MG cells were inhibited.Conclusion:dl-THP can inhibit the proliferation of U251 MG cells in vitro, and promote apoptosis.The mechanism of promoting apoptosis may be related to the activation of P53 and stability expression of its downstream factor p21.The second experiment:The anti-tumor effects study of dl-THP on tumor bearing mice.Objective: To observe theanti malignant glioma effects of dl-THP on tumor bearing mice and the influence of protein p21 and p53 expression.Methods:We establish theanimal model of malignant glioma nude mice by growing U251 MG cells in BALB/C-nu nude mice subcutaneous.the establishment of a. Continuous administration of dl-THP(1mg/Kg/day, 2mg/Kg/day) for 10 days,stripping off the tumor body,recording the weight and volume, calculating the tumor inhibition rates,and observing survival time of model mice. Immune histochemical method and Western blotting were adopted to measure the protein expression of p21 and p53 in tumor tissues after dl-THP treatment.Results:(1)the tumor inhibition rate of dl-THP in the intervention group were 54.2%(dl-THP 1mg/Kg/day) and 65.9%(dl-THP 2mg/Kg/day), and have a significant difference compared with the control group(P<0.05);(2) The longest survival time was 34 d afterdl-THP intervention;(3)The protein expression of p53 and p21 in the tumor tissue up regulation(P<0.05).Conclusion: dl-THP can significantly inhibit the growth of tumor tissue, prolong the survival time of tumor bearing mice,the possible mechanism may be related to up regulate the protein expression of p21 and p53 in tumor tissues,which promoting malignant glioma apoptosis.Part 3 The study of Simulated microgravity on the influence of human malignant glioma U251 MG cells apoptosis and related molecular mechanismsObjective: we have observed the simulated microgravity on the influence of U251 MG proliferation, cell cycle and apoptosis and promote the mechanism of apoptosisMethods: simulated microgravity intervention U251 MG cells in different time(12h, 24 h, 48 h, 72h), by using CCK 8 to detect U251 MG cells proliferation, the influence of flow cytometry to detect the cell cycle change, transmission electron microscopy 、 flow cytometry and tunel to observe cell apoptosis; fluorescent glass protein chip screening apoptosis-associated factors, and using Western blot method and real time PCR detection of simulated microgravity on apoptosis related protein IGFBP-2 and p21 protein and m RNA expression.’Results:(1) After the simulated microgravity intervention in different time of 12 h, 24 h, 48 h, 72 h, U251 MG cells proliferation significantly suppressed which have time dependence as well;(2) U251 MG cell cycle distribution changed significantly(P<0.05), and with the increase of simulated microgravity intervention time, gradually change by the arrest of S phase to G0/G1 phase arrest(3)The apoptosis rate of U251 MG cells increased with the time of intervention(P<0.05); and the emergence of structural and morphological changed.(4) The expression of p21 protein and m RNA of U251 MG cells regulate increasedwhile the expression of IGFBP-2 regulatedecrease(P<0.05).Conclusion: simulated microgravity can inhibit the proliferation of U251 MG, block the cell cycle andmotivate its apoptosis. The mechanism of apoptosis may be related toabnormal expression of p21 and IGFBP-2.