Objective To observe the effects of butylphthalide on the expression of autophagy-related protein and mRNA in l-meth-yl-4-phenyl-pyridiniumion (MPP+)-induced SH-SY5Y cells, and to explore the protective effect and possible mechanism of butylphthalide to the cell model of Parkinson's disease. Methods The SH-SY5Y cells were divided into control group (A), MPP+group (B), rapamycin pre-treated+MPP+group (C) and Butylphthalide pretreated+MPP+group (D). The relative viability of SH-SY5Y cells induced by MPP+was measured with MTT assay, the morphology of SH-SY5Y cells was observed. The expression of microtubule associated protein 1 light chain 3 (LC3)-II/I and Beclin 1 protein was detected by Western blotting. And the expression of LC3-II/I and Beclin 1 mRNA were assayed by re-al-time quantitative reverse transcription-PCR (RT-PCR). Results The viability rates of cells were significantly lower in group B than in group A (t=20.270, P<0.001), and were significantly higher in groups C and D than in group B (t>8.770, P<0.001), however, there was no significantly difference between groups C and D (t=2.270, P=0.064). The expression of LC3-II/I and Beclin 1 was higher in group B than in group A (t>6.647, P<0.01), and was higher in groups C and D than in group B (t>3.630, P<0.01), however, there was no significantly differ-ence between groups C and D (t<2.238, P≥0.05). Conclusion Butylphthalide could prevent the injury of SH-SY5Y cells induced by MPP+, which may affect Parkinson's disease by inducing autophagy.

Objective To evaluate the mutagenicity of hydrolysate of Meretrix meretrix Linnaeus soft tissue, so as to provide experimental basis for its exploitation.Methods Three mutagenicity tests were used to evaluate the mutagenic effects, including Ames test, CHL chromosome aberration assay and bone marrow micronucleus assay in mice.Results In Ames test, the revertant colonies numbers in each group were twice less than the numbers of spontaneous revertant colo-nies, five bacterial strains showed negative results with or without S9 activation, and the result of Ames test was negative. The CHL chromosome aberration assay and bone marrow micronucleus assay showed that the chromosome aberration rate and micronucleus rate of each dose group showed no significant difference compared with the negative control group, respec-tively ( P>0.05) .Conclusions Under this condition, the results show that all of the Ames test, chromosome aberration assay and bone marrow micronucleus assay are negative, and no mutagenicity is observed in the hydrolysate of Meretrix mer-etrix Linnaeus soft tissue.

ObjectiveTo explore the relationship of trefoil factor family 3 (TFF3),vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1α in gastric cancer SGC-7901 cells under hypoxic condition and try to investigate the mechanism of TFF3 in the genesis and development of gastric cancer. Methods The hypoxic model of gastric cancer SGC-7901 cell was induced by CoCl2 Gastric cancer cell line SGC-7901 cells were transfccted with pU6-siTFF3 plasmid which carrying RNAi targeted to human TFF3 and pU6-mock.Puromycin was selected as screening medicine.The stable and specific TFF3 inhibited gastric cancer cell line was established. Gastric cancer cell line SGC-7901 and TFF3 RNAi targeted gastric cancer cell line SGC 7901 were cultured under hypoxic condition and normoxic condition. The expression of TFF3,VEGF and HIF-1a at protein and mRNA level were detected by RT-PCR,Western blot and ELISA assay.The distribution and expression of TFF3 and HIF-1α in gastric cancer cell line SGC-7901 cells uuder normoxia and hypoxic condition were determined with immunofluorescence staining.Results The expressions of HIF-1a,TFF3 and VEGF in gastric cancer SGC-7901 cell increased under CoCl2 induced hypoxic condition (33.4 =1.8,14.8 ± 1.1 and 15.1 ± 1.2,respectively). Under hypoxie condition,the expression of VEGF and HIF-1α protein reduced in stable TFF3 RNAi SGC-7901cells.Conclusion TFF3 mediated the regulation of VEGF and HIF-1α expression under hypoxic condition.TFF3might be a potential anti-angiogenic target in gastric cancer treatment.

Objective To analyze the protective effects ofbutylphthalide(NBP) on apoptosis factors (p-JNK,Fas and FasL) of death receptor pathway in JNK pathway of cell model of Parkinson's disease (PD).Methods SH-SY5Y cell apoptosis model induced by MPP + was established in vitro.The cells were divided into four groups:normal control group,SHSYSY cells were treated with complete medium without drug intervention;MPP+ group,1 mmol·L-1 MPP+ was added into the cells;NBP+ MPP+ group,the cells were pretreated with 10 mol·L-1 NBP for 3 h and added with 1 mmol·L-1 MPP+;SP600125 + MPP+ group,the cells were cultured with 10 mol·L-1 JNK inhibitor SP600125 pretreatment for 3 h and 1 mmol·L-1 MPP+ was added.The proliferative potentiality of SH-SY5Y cells induced by MPP+ was measured by MTT.The apoptotic rate was analyzed by Annexin-V/PI (FCM).The morphology of SH-SY5Y cells was observed by inverted phase contrast microscope.The expression of apoptotic protein p-JNK,Fas,FasL was detected by Western blotting.Results The cell proliferative potentiality in the MPP+ group (49.30 ± 2.07)％ was significantly lower than that of the normal control group (100.00 ±0.00)％ (P ＜ 0.05).The cell proliferative potentiality in NBP + MPP + group and SP600125 + MPP + group were (71.90 ±2.10) ％ and (76.40 ± 2.80) ％,which was significantly higher than that of the MPP + group (P ＜ 0.05).Apoptosis rate in the MPP + group (32.27 ± 2.26) ％ was significantly higher than that of the normal control group (10.63 ± 2.07) ％ (P ＜ 0.05).The apoptosis rate in the NBP + MPP + group and SP600125 + MPP+ group were (21.13 ± 3.63) ％ and (19.15 ± 2.63) ％,and the apoptosis rate was significantly lower than that in the MPP+ group(P ＜0.05).The protein expression levels of p-JNK,Fas and FasL were significandy lower in NBP + MPP+ group and SP600125 + MPP+ group than that in the MPP+ group (P ＜0.05).Conclusion Butylphthalide can protect the injury of SH-SY5Y cells induced by MPP+.The mechanism of butylphthalide inhibiting apoptosis may be achieved through regulating p-JNK,Fas and FasL protein expression of death receptor pathway in JNK pathway and inhibiting the cell apoptosis.

BACKGROUND:Bone marrow mesenchymal stem cells have the potential of self-proliferation and multi-directional differentiation, while mesenchymal stem cells are few in adult bone marrow. In vitro purification, amplification and osteoinduction are very important for the research of bone tissue engineering. OBJECTIVE:To establish a simple and reliable in vitro cultivation and identification system of adult bone marrow mesenchymal stem cells, and to induce the mesenchymal stem cells to differentiate into osteoblasts. METHODS:Bone marrow were extracted from adult anterior superior iliac, the density gradient centrifugation and adhesion method were used to isolate, purify, culture and amplify the bone marrow mesenchymal stem cells. Osteogenic medium was prepared by mixing appropriate amount of dexamethasone,β-glycerophosphate and ascorbic acid C. The cells were divided into osteoinduction group and blank control group for observation.
RESULTS AND CONCLUSION:Adult bone marrow mesenchymal stem cells were in typical long spindle-shape. The cells grew into rapid proliferation phase at 8-11 days and the growth curve was S-shape. CD44 and CD90 were in positive expression, while CD34 and CD45 were negative. The alkaline phosphatase activity was increased with culturing time prolonging, and reached the summit at the 12th day. The alkaline phosphatase activities of osteoinduction group were higher than those in the blank control group at different time points. These results suggested that in vitro cultivation, identification and osteoinduction system could obtain mesenchymal stem cells with high purity and good osteogenic differentiation capacity.

Objective To explore the effects of butylphthalide on the apoptosis of SH-SY5Y cells induced by the 1-methyl-4-phenylpyridinium iodide (MPP+). Methods SH-SY5Y cells were cultured with butylphthalide in the dosage of 1 µmol/L, 10 µmol/L and 20 µmol/L for 2 hours, and with MPP+ for 24 hours. The viability of cells was measured with trypan blue. The mitochondrial membrane potential was detected with Rhodamine 123. The content of P53 protein was detected with ELISA. The expression of P53 and cytochrome C (CytC) were detected with Western blotting. Results The viability of SH-SY5Y cells increased with the dosage of butylphthalide (P<0.05), while the mitochondrial membrane potential and the expression of P53 and CytC decreased (P<0.05). Conclusion Butylphthalide can prevents the SH-SY5Y cells from the injury induced by MPP+, which may associate with the inhibition of apoptosis through the expression of P53 and CytC.

Objective To study the influencing factors and quality control of chromosome preparation during chromosome aberration test in vitro,and to summarize and analyze the method and key points of successful preparation of chromosome specimens in vitro. Methods Chinese hamster lung cells(CHL)were used for cell culture and chromosome preparation. Mitomycin and cyclophosphamide were used as positive mutagens. After routine hypotonic treatment,fixation, and squash preparation, finally, to read the film under the microscope. Results The CHL chromosome aberration test showed that both the chromosome aberration rates of mitomycin- and cyclophosphamide-treated cells were significantly increased(>20%),while the aberration rates in the negative control group were less than 5%, either with and without metabolic activation. The success rate was high and the prepared chromosomes were well dispersed with a moderate length. Conclusions Many factors can affect the specimen preparation in chromosome aberration test. Every step is very important,and it should be strictly following the operating procedures. It is of importance to grasp the principle of each step and to operate carefully,patiently and scientifically in order to prepare good specimens.