[Synchronization isolation method for multiple types of cells from mouse liver].

Objective: To explore a simple and feasible method for the isolation and purification of hepatocytes, hepatic stellate cells (HSC), and lymphocytes from mice. Methods: The cell suspension was obtained from male C57bl/6 mice by hepatic perfusion through the portal vein digestion method and then isolated and purified by discontinuous Percoll gradient centrifugation. Trypan blue exclusion was used to determine cell viability. Glycogen staining, cytokeratin 18, and transmission electron microscopy were used to identify hepatic cells. Immunofluorescence was used to detect α-smooth muscle actin combined with desmin in HSCs. Flow cytometry was used to analyze lymphocyte subsets in the liver. Results: After isolation and purification, about 2.7×10(7) hepatocytes, 5.7×10(5) HSCS, and 4.6×106 hepatic mononuclear cells were obtained from the liver of mice with a body weight of about 22g. The cell survival rate in each group was > 95%. Hepatocytes were apparent in glycogen deposited purple-red granules and cytokeratin 18. Electron microscopy showed that there were abundant organelles in hepatocytes and tight junctions between cells. HSC had expressed α-smooth muscle actin and desmin. Flow cytometry showed hepatic mononuclear cells, including lymphocyte subsets such as CD4, CD8, NKs, and NKTs. Conclusion: The hepatic perfusion through the portal vein digestion method can isolate multiple primary cells from the liver of mice at once and has the features of simplicity and efficiency.

Induction of apoptosis in HepG2 cells by solanine and Bcl-2 protein.

The nightshade (Solanum nigrum Linn.) has been widely used in Chinese traditional medicine as a remedy for the treatment of digestive system cancer. The anti-tumor activity of solanine, a steroid alkaloid isolated from the nightshade has been demonstrated. To observe the effect of anti-tumor and mechanism of solanine. The MTT assay was used to evaluate the IC(50) on the three digestive system tumor cell lines. The effect on the morphology was observed with a laser confocal microscopy; the rate of apoptosis and the cell cycle were measured using flow cytometry (FCM); the expression of Bcl-2 protein was measured by Western blot. The results show that the IC(50) for HepG(2), SGC-7901, and LS-174 were 14.47, >50, and >50 microg/ml, respectively; the morphology of cells in the negative control was normal; for the treated groups, typical signs for apoptosis were found. The rate of apoptosis in HepG(2) cells induced by solanine was found to be 6.0, 14.4, 17.3, 18.9, and 32.2%, respectively. Observation of the cell cycle showed that cells in the G(2)/M phases disappeared while the number of cells in the S phase increased significantly for treated groups. Western blot showed that solanine decreased the expression of Bcl-2 protein. Therefore, the target of solanine in inducing apoptosis in HepG(2) cells seems to be mediated by the inhibition in the expression of Bcl-2 protein.

[The role of substance P and somatostatin in acupuncture and moxibustion-induced postsynaptic inhibition].

AIM OF INVESTIGATION: Our previous work indicated that acupuncture and moxibustion (heating acupoints with a special lamp) could produce inhibitory effects on the tail flick reflex and the nociceptive response of dorsal horn neurons. In the present study the role of somatostatin (SS) and substance P (SP) in acupuncture and moxibustion-induced postsynaptic inhibition was further investigated. METHODS: The experiments were performed on male adult Wistar rats. The antidromic action potential (AAP) induced by cervical cord stimulation was extracellulary recorded at lumbar cord (L3-4). The latency of AAP was used to represent the excitability of postsynaptic projecting neurons. The effects of acupuncture (0.5 ms, 3.3 Hz, 2 mA) for five minutes or moxibustion (the temperature up to 45-46 degrees C) for six minutes at Huantiao points on the latency of AAP were observed. And then the effects of topical administration of SS or SP antiserum on either acupuncture or moxibustion-induced postsynaptic inhibition were observed. RESULTS: The latency of AAP was markedly prolonged by acupuncture and moxibustion. The maximal prolongation was 0.196 +/- 0.071 ms (n = 12, P < 0.02) and 0.176 +/- 0.062 ms (n = 11, P < 0.02) respectively. After topical administration of SS antiserum (1:40, 10 microliters), the latency of AAP was slightly prolonged by either acupuncture or moxibustion. The maximal prolongation (0.041 +/- 0.029 ms and 0.016 +/- 0.020 ms) was significantly reduced by SS antiserum (P < 0.05). While pretreated with SP antiserum, the latency was still prolonged by moxibustion (0.142 +/- 0.067 ms), but not by acupuncture (-0.003 +/- 0.046 ms). CONCLUSION: It is referred that postsynaptic inhibition may be involved in both acupuncture analgesia and moxibustion analgesia. The former is predominately mediated by SP and partially by SS, while the latter mainly by SS but not by SP.

Dual action of substance P in nociception and pain modulation at spinal level.

In the present study, the activation of the nociceptive dorsal horn neurons in rats by iontophoretic substance P (SP) has been observed. Thus, the role of SP in spinal nociception is further identified. In addition, the inhibitory effects of iontophoretic SP on nociceptive response (C response) of dorsal horn neurons can also be observed even in rats that have undergone dorsal half transection of spinal cord. These inhibitory effects can be partially blocked by pretreatment with iontophoretic bicuculline but not by naloxone. It indicates that the SP-induced inhibitory effects on the nociceptive response may be mainly mediated by the presynaptic inhibition in which gamma-aminobutyric acid (GABA) may be involved. In view of the fact that the inhibitory effect of stimulation of nucleus raphe magnus (NRM) on the nociceptive response of dorsal horn neurons in rats depleted of 5-HT with parachlorophenylalanine (pCPA) can be significantly blocked by iontophoretic SP-antagonist, it is supposed that SP may be involved in descending modulation on spinal pain transmission.