| PrefaceIn 1975, methionine enkephalin(MENK) with endogenous morphine-like activity was found in extract from pig brain. Later study showed that endogenous aglucone of opioid receptors as a natural material derived from adrenal gland prehormone and proenkephalin could combine with opioid receptors to generate stabilization and analgesic effect on human and animals. Recent study showed that MENK had regulating function in neuroendocrine system. Immunoregulation consists of many functions as to increase the activities of Tlymphocytes, NK cells and macrophages in immune system and to increase the releasing of IL-2,γ-interferon and etc. MENK could also inhibit the growth of tumor cells. Some researches had proved that MENK could be used single or combined with other cytokines. MENK, combined with IFN-y could strengthen the function of immune cells significantly. MENK also had a conspicuous therapeutic effect on cancer and AIDS. The number of CD4+T cells were increased in patients'peripheral blood.Opioid receptor has several hypotypes includingμ(mu),δ(delta),κ(kappa). Some sudies had proved that in different situations MENK could transmit signals into cells through varied channels. The main channel of signal transmission can be changed and generate different biological effect on different cells especially the dose-effect relationship when MENK combine with different receptors in different concentration. CD4+T cells are important cell subgroup in body specificity cell immune. Some reports showed that MENK could improve the cytoactive of CD4+T cells, but the definite dose-effect relationship had not been proved.In the study, we used flow cytometry to count the number of CD4+T cells, and RT-PCR to detect the mRNA content in CD4 molecule, in order to find the best concentration and effect of MENK to stimulate the proliferation of CD4+T cells both in vivo and in vitro. The investigation on the effects of MENK to stimulate the proliferation of CD4+T cells can provide theory and experiment basis for clinical application.Materials and Methods1. The CD4+T cells analysis by flow cytometry.CD4+T cells, separated aseptically from mice spleens, stimulated by range of concentration MENK both in vivo and in vitro and counted by flow cytometry.T-test was done between the data of experimental groups and the data of control group. Statistical analysis was done by SPSS 11.5 software for all data.2. The CD4 molecule confirmation at mRNA level by RT-PCRThe mRNA of CD4 molecule, enriched by magnetic bead CD4+T cells from lymphocyte which were separated aseptically from mice spleens, stimulated by range of MENK both in vivo and in vitro, was detected by RT-PCR. T-test was done between the data of experimental groups and the data of control group. Statistical analysis was done by SPSS11.5 software for all data.Results1.MENK (at the concentration of 10-9M to 10-12M in vitro and 200mg/kg in vivo) could significantly promote the proliferation of CD4+T cells (p＜0.05); however MENK (at the concentration of 10-5M in vitro) inhibited the proliferation of CD4+T cells (p＜0.05); MENK (at the concentration of 10-6M to 10-8M and 10-13M to 10-14M in vitro, as well as 50mg/kg and 100mg/kg in vivo) had no effect (p＞0.05),2.MENK (at the concentration of 10-9M to 10-12M in vitro and 200mg/kg in vivo) could significantly promote the transcription of mRNA on CD4 molecule (p＜0.05); however MENK (at the concentration of 10-5M in vitro) inhibited the transcription of mRNA on CD4 molecule (p＜0.05); MENK (at the concentration of 10-6M to 10-8M and 10-13M to 10-14M in vitro, as well as 50mg/kg and100mg/kg in vivo) had no effect (p＞0.05).Conclusion1.MENK (at the concentration of 10-9M to 10-12M in vitro) could significantly promote the proliferation of CD4+T cells. MENK at the higher concentration could inhibit the proliferation, while MENK at the lower concentration had no effect.2.MENK (at the suitable concentration of 200mg/kg in vivo) could promote the proliferation of CD4+T cells.3.Conclusion above was confirmed at mRNA level.
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