Regulation Of Rapamycin In The Process Of RAW264.7Cells Infected By BCG

Mycobacterium tuberculosis(MTB) caused TB, it is a chronic infectious disease of zoonotic. MTB is a typical cellular parasite, MTB interaction with host macrophages incidence of tuberculosis and the pathological development plays an important role in many intracellular signaling molecules have been shown to participate in this pathogen-host interaction. Mammalian target of rapamycin (mTOR) signaling pathways regulating cell growth and proliferation is a key signaling pathways. In the immune system, mTOR signaling pathway in stimulating and regulating the inflammatory response also plays an important role. mTOR inhibitor rapamycin can affect LPS-induced apoptosis in macrophages NO production or macrophages through different mechanisms, but on rapamycin in BCG infection of macrophages and cells for NO regulation of apoptosis has not been reported.1. In order of fully experimental materials, we were cultured BCG in solid culture and liquid culture. During cultured in liquid, we add the glycerin and Tween-80in two different culture media. We found that in liquid culture added Tween-80even more suitable for the growth of BCG, and can provide a good foundation for the subsequent experiments. In the count of BCG, we took three different counting methods:turbidimetry, acid-fast staining and FDA staining; and count the results of these three methods were statistically and compared. We found that FDA staining and acid-fast staining is more suitable for BCG counts, especially FDA staining can be more accurate count of the number of viable cells of BCG2. We studied the effects of rapamycin in the course of BCG infection in RAW264.7cells to produce the amount of NO and inducible nitric oxide synthase (iNOS) mRNA and protein expression in LPS-stimulated simultaneously set up the control group. The results showed that, compared with the control group, BCG, LPS stimulation of6,12and24h can produce a significant increase in the levels and the expression of NO, iNOS mRNA and protein in RAW264.7cells (P<0.01).Compared with the group without adding rapamycin, BCG infection during6and12h,10nmol/L rapamycin can significantly inhibit the expression of NO and iNOS in RAW264.7cells (P><0.01), however, at24h rapamycin inhibition of stimulated NO production was not significant (P>0.05), but the same can significantly inhibit the expression of iNOS (P<0.01). LPS stimulation of6and24h after,10nmol/L rapamycin can significantly inhibited NO production in RAW264.7cells (P<0.01); during12h, significantly inhibited NO production (P<0.05). When LPS stimulation rapamycin inhibited the expression of iNOS6h when the difference was not significant (P>0.05),12and24h, the difference was significant (P<0.01).3. The effects of rapamycin BCG infection in RAW264.7cells on apoptosis.The results showed that within1nmol/L-lμmol/L concentration range of10nmol/L rapamycin inhibition of apoptosis most significant (P<0.01). Flow cytometry showed the impact of rapamycin on apoptosis BCG infection by promoting6h at24h and gradually transformed into inhibition when; while the LPS-treated group, rapamycin on cells wither death was no significant difference (P>0.05). By detecting NF-κB activation levels found after BCG and LPS treatment can significantly improve the activity of NF-κB (P<0.01or P<0.05), and rapamycin on BCG treatment groups NF-κB activity6h when the inhibitory effect of promoting a gradual turn at24h, but in the LPS-treated group has been doing to promote the role of NF-kB activity (P<0.01). Further using ELISA method to detect the cells produce TNF-a levels, the results show that when the6h rapamycin promote the generation (P<0.05or P<0.01) TNF-a stimulation after BCG and LPS infection, but12and24h there was no significant difference (P>0.05). In dealing with24h of apoptosis-related protein Caspase-3analysis found that rapamycin after BCG infection can inhibit Caspase-3activity, mRNA and protein expression (P<0.05or P<0.01); But after LPS stimulation inhibition was not significant (P>0.05). In addition, rapamycin also significantly inhibited the BCG infection after24h in the G1phase of the cell cycle the cell occupied by the ratio (P<0.01).