| OBJECTIVE:By detecting the expression of Bcl-2 and Bad protein to investigate the effect of Helicobacter pylori(Hp) on human gastric cancer cell line SGC-7901 proliferation,apoptosis and understand the pathogenic mechanism of Hp further。METHODS:Human gastric cancer cell line SGC-7901 was incubated with different concentrations(104,105,106,107cfu/ml) of Hp for u. The effect of Hp on cell proliferation was detected by MTT assay.The expression of Bcl-2 and Bad protein was investigated by immunohistochemistry.RESULT:1.Hp culture and freezing keeping method. (1)colonial morphology Hp colony was translucent and like needle tip.Its diameter was 1-2mm.when we inoculate considerable strain,the colony can mix together to form a tier of translucent lawn.(2)bacterial smear Hp was Gram-negative bacteria,comma,benting like S or short rod. When cultured more than five days,Hp can become globular.(3)Biochemistry accredit of Hp typical bending Hp rapid urease test,oxidase test and catalase test were positive,but the globular Hp biochemistry test appearance weakly positive.(4) freezing keeping method In short time using normal saline to keep Hp strain is better,but it can not freeze thawing the strain repeatedly.2.cellular morphology Normal SGC-7901 cell paste to the board bottom tightly,fusiform or polygon,very clarity and the refraction was strong,part of the cell microarchitecture can be seen.But after 12 hours co-cultured with Hp,the cell shrink, cytoplasm condense and taking off from the board bottom.with the time prolong,cell occurring vacuole pathological changes and more toxic granulation can be seen.It is very obviously at 107cfu/ml group. After 48h, there were part of cell disruption at106,107cfu/ml groups,forming fragment.3.MTT assay The OD of cell in each group was all raised with the time prolong.After 12h and 24h there is no significant difference of cell OD between SGC-7901 co-cultured with Hp at low concentrations (104,105cfu/ml) and control group (P>0.05),Co-culture 48h, the OD of low concentrations (104,105cfu/ml) is higher than control group (P< 0.01);Co-culture with high concentrations(106,107cfu/ml) groups,at 12h,the OD of 107cfu/ml group is lower than control group (P< 0.05),Co-culture 24h, the OD of high concentrations(106,107cfu/ml) is lower than control group (P< 0.01), Co-culture 48h, the OD of high concentrations(106,107cfu/ml) groups is lower than control group (P< 0.01).4. immunohistochemistry result (1) Expression of Bcl-2 protein Co-culture 12h, there is no significant difference of Bcl-2 protein expression at SGC-7901 co-cultured with Hp groups compared with control group (P>0.05) Co-culture 48h, there is significant difference of Bcl-2 protein expression at each groups compared with control group(P<0.01). (2) Co-culture 12h, there is no significant difference of Bad protein expression at 104,105,106 groups compared with control group (P>0.05),but that at 107cfu/ml group is higher than control group (P < 0.05). Co-culture 48h, there is significant difference of Bad protein expression at each groups compared with control group(P<0.01). It shows that at low concentration groups is lower than control group,that at high concentration groups is higher than control group.CONCLUSION:1.Using normal saline to keep Hp strain was better in short term. Spherical body of Hp may play important part in Hp transmission and post-treatment recurrence.2.Useing Hp infected SGC-7901 in vitro,without of the inflammatory medium and cytokine in vivo. SGC-7901 is easy to culture, growing fast and easy to transfer of culture,making experiments repeatability raise.3.Different Hp concentrations had different influence on cell proliferation and apoptosis.It may be concerned with Bcl-2 and Bad protein.(1) Hp at low concentrations led to cell proliferation.The expression of Bcl-2 protein increased.And Bad expression decreased.(2) Hp at high concentrations cause cell vacuolar degeneration, inhibiting cell proliferation, promoting cell apoptosis. The expression of Bcl-2 protein decreased and the expression of Bad protein increased.To demonstrate that cell proliferation accompanied with apoptosis,and this effect in high concentration group can be showed in short time, more obviously more time. |
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