Knockdown of TMPRSS3 inhibits gastric cancer cell proliferation, invasion and EMT via regulation of the ERK1/2 and PI3K/Akt pathways.

The transmembrane protease, serine 3 (TMPRSS3), a member of the type II transmembrane serine protease family, plays an important role in mediating tissue development, homeostasis and various biological processes. Recently, TMPRSS3 has been reported to be involved in cancer progression. However, the role of TMPRSS3 in gastric cancer (GC) remains largely unknown. In this study, we found that TMPRSS3 was highly expressed in GC tissues and cell lines. Knockdown of TMPRSS3 inhibited GC cell proliferation, invasion and epithelial-mesenchymal transition (EMT) in vitro as well as suppressed GC cell growth and dissemination in vivo. These inhibitory effects were mediated by regulation of the ERK1/2 signaling pathway. Moreover, TMPRSS3-mediated ERK1/2 activation was dependent on the PI3K/Akt pathway. In conclusion, TMPRSS3 contributed to GC progression via activation of the PI3K/Akt/ERK signaling pathway and might act as a therapeutic target for GC treatment.

Up-regulation of hypoxia inducible factor-1α by cobalt chloride correlates with proliferation and apoptosis in PC-2 cells.

BACKGROUND: The exact mechanism of the effects of hypoxia on the proliferation and apoptosis in carcinoma cells is still conflicting. This study investigated the variation of hypoxia-inducible factor-1α(HIF-1α) expression and the apoptosis effect of hypoxia stimulated by cobalt chloride (CoCl(2)) in pancreatic cancer PC-2 cells. METHODS: PC-2 cells were cultured with different concentration (50-200 µmol/L) of CoCl(2) after 24-120 hours to simulate hypoxia in vitro. The proliferation of PC-2 cells was examined by MTT assay. The cellular morphology of PC-2 cells were observed by light inverted microscope and transmission electron microscope(EM). The expression of HIF-1α on mRNA and protein level was measured by semi-quantitative RT-PCR and Western blot analysis. Apoptosis of PC-2 cells were demonstrated by flow cytometry with Annexin V-FITC/PI double staining. RESULTS: MTT assay showed that the proliferation of PC-2 cells were stimulated in the first 72 h, while after treated over 72 h, a dose- dependent inhibition of cell growth could be observed. By using transmission electron microscope, swollen chondrosomes, accumulated chromatin under the nuclear membrane and apoptosis bodies were observed. Flow cytometer(FCM) analysis showed the apoptosis rate was correlated with the dosage of CoCl(2). RT-PCR and Western blot analysis indicated that hypoxia could up-regulate the expression of HIF-1α on both mRNA and protein levels. CONCLUSION: Hypoxic microenvironment stimulated by CoCl(2) could effectively induce apoptosis and influence cell proliferation in PC-2 cells, the mechanism could be related to up-expression of HIF-1α.

In vitro and in vivo antitumor activity of Scutellaria barbate extract on murine liver cancer.

In the present study, we investigated the in vitro and in vivo antitumor effects of crude extract of Scutellaria Barbate (CE-SB) on mouse hepatoma H22 cells. The MTT assay was used to determine the growth inhibition of H22 cells in vitro. The in vivo therapeutic effects of CE-SB were determined using H22 tumor bearing mice. Besides, the body weight, tumor weight, thymus index and spleen index of H22 bearing mice were also measured. The tumor inhibitory rate (IR) was calculated according to the mean weight of tumor (MWT). The phagocytotic function of macrophages was examined by observing peritoneal macrophages phagocytize chicken RBC. The results showed that CE-SB could inhibit the growth of hepatoma H22 Cells in vitro and in vivo. Furthermore, CE-SB could improve immune function of H22 tumor bearing mice. Together these results indicate that CE-SB has antitumor activity and seems to be safe and effective for the use of anti-tumor therapy.

[Role of vascular endothelial growth factor in rats with severe acute pancreatitis].

OBJECTIVE: To investigate the role of vascular endothelial growth factor (VEGF) in the pathogenesis of severe acute pancreatitis (SAP) in rats. METHODS: Sixty-four male SD rats were randomly divided into control group and SAP group, and in the latter group, SAP was induced by retrograde injection of 5% sodium taurocholate in the pancreaticobiliary duct. The rats were sacrificed at 1, 3, 6 and 12 h after the operation, and the severity of pancreatitis was assessed according to histological scoring. The serum levels of VEGF were examined with enzyme-linked immunosorbent assay, and the expression of VEGF in the pancreatic tissues was measured by SP immunohistochemistry. Another 30 SD rats were randomized into the control group, SAP group and SAP+recombinant rat VEGF injection group, and the vascular permeability of the pancreatic microcirculation was determined by Evans Blue leakage test. RESULTS: At each of the time points for measurement, both the serum VEGF level and scores of pancreatic tissue injury were significantly higher in SAP group than in the control group (P<0.05). Compared with the control group, the expressions of VEGF in the pancreatic tissues of SAP group were significantly up-regulated following the operation (P<0.05). The vascular permeability of the pancreatic microcirculation significantly increased after the onset of SAP, and injection of recombinant rat VEGF significantly increased the leakage rate of Evans Blue. CONCLUSION: VEGF may play an important role in the pathogenesis of pancreatitis and in causing edema and hemorrhage in SAP, and the level of serum VEGF may reflect the severity of pancreatic injury.

[Protective effects of captopril against lung injury in rats with severe acute pancreatitis].

OBJECTIVE: To investigate the protective effects of captopril against lung injury in a rat model of severe acute pancreatitis (SAP). METHODS: Seventy-two male SD rats were randomized into sham-operated group (SO group), SAP group and captopril intervention group (CAP group). Serum amylase and myeloperoxidase (MPO) activity in the lung tissue were examined at 1, 6 and 12 h after the operation. TNF-α and AngII in the lung tissue were detected by ELISA, and the histopathological changes of the pancreas and lung were observed microscopically. RESULTS: The MPO activity , which was similar between SAP group and CAP group at 1 h, were significantly lowered in CAP group at 6 and 12 h (P<0.05). Serum amylase level and the levels of TNF-α and AngII in the lung tissue homogenate were all reduced significantly in CAP group as compared to those in SAP group (P<0.01). The pathological injury of the lung was obviously lessened in CAP group in comparison with that in SAP group. CONCLUSION: Captopril can ameliorate SAP-induced lung injury in rats.

Notch1 regulates the growth of human colon cancers.

BACKGROUND: The aberrant activation of the Notch signaling has been associated with the development of colon cancers. However, the role of Notch1 in the pathogenesis of colon cancers is poorly understood. METHODS: The expression of Notch1 in colon cancer tissues and nontumor tissues and in colon cancer cell lines was examined by Western blot analysis and immunohistochemistry. The impact of small interfering RNA (siRNA)-mediated Notch1 knockdown or Notch1 intracellular domain (NICD)-based transgene-induced Notch1 overexpression on the proliferation, cell cycling, apoptosis, colony formation, and tumorsphere formation in vitro and the development and growth of implanted tumors in vivo was characterized. RESULTS: Notch1 was overexpressed in colon cancer tissues, and the levels of Notch1 expression in different types of colon cancers were associated with the pathologic grade, progression, and metastasis of colon cancers. Furthermore, knockdown of Notch1 significantly inhibited the proliferation, colony formation, and tumorsphere formation of SW480 and HT-29 cells, induced apoptosis and cell cycle arrest at G0/G1 phase, and mitigated the development and growth of implanted colon cancers in vivo. In contrast, Notch1 overexpression promoted the proliferation, colony formation, cell cycling, and tumorsphere formation of colon cancer cells in vitro and the development and growth of implanted colon cancers in vivo, but it inhibited spontaneous apoptosis. CONCLUSIONS: The current results indicated that Notch1 signaling positively regulates the growth of colon cancers. Conceivably, the modulation of Notch1-related signaling may be a promising therapy for human colon cancers.

[Inhibitory effects of Scutellaria barbate extracts on diethylnitrosamine-induced hepatocarcinoma in rats].

OBJECTIVE: To investigate the inhibitory effects of Scutellaria barbate extracts on diethylnitrosamine-induced hepatocarcinoma in rats. METHODS: Hepatocarcinoma model rats were induced by diethylnitrosamine (DEN). Sixty SD male rats were randomly divided into 4 groups: normal control group, hepatocarcinoma model group, ESB of high dose group and ESB of low dose group. All rats were killed in the 18th week, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), alkaline phosphatase (ALP), gamma-glutamyltransferase (gamma-GT) and alpha-L-fucosidase (AFU) in serum were measured by biochemical examinations; Hematoxy and eosin (HE) methods were used to examine the changes of liver pathology. RESULTS: The levels of ALT, AST, TBIL, ALP, gamma-GT, AFU in hepatocarcinoma model group and ESB groups were higher than that of control group (P < 0.05). ESB could relieve hepatic injures. The levels of liver function indexes in ESB groups were lower than that of model group. Histological examination demonstrated that the number of liver cancer nodus in ESB groups were lower than that of model group. Furthermore, ESB could attenuate the grade of cancer cell differentiation. CONCLUSION: ESB could inhibit experimental hepatocarcinoma and relieve hepatic injures in rats.

Inhibiting gastric cancer-associated angiogenesis by CIAPIN1 siRNA.

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising target for cancer therapy. We characterized the effects of selective CIAPIN1 inhibition on the angiogenesis gastric cancer cell line SGC7901 by stable transfection of CIAPIN1 siRNA. Our study has been shown that CIAPIN1 play the determined role in tumor growth and multidrug resistance. The conditioned media obtained from SGC7901 treated with CIAPIN1 siRNA suppressed in vitro the proliferation, migration and tube formation of human umbilical vein endothelial cells compared with untransfected cells or cells transfected with control vector alone. Furthermore, the stable transfection of CIAPIN1 siRNA inhibited in vivo tumorigenicity and angiogenesis. Our findings support that selective inhibition of CIAPIN1 alone plays an instrumental role on gastric cancer associated angiogenesis.

Matrine induces apoptosis in gastric carcinoma cells via alteration of Fas/FasL and activation of caspase-3.

AIM OF THE STUDY: Matrine, an alkaloid purified from the chinese herb Sophora flavescens Ait, is well known to possess activities including anti-inflammation, anti-fibrotic and anticancer. In this study, the mechanism of matrine inducing the apoptosis of gastric carcinoma cells was investigated. MATERIALS AND METHODS: Proliferation of SGC-7901 cells was examined by MTT assay. Cellular morphology was observed under transmission electron microscope. Flow cytometry (FCM) was used to observe the apoptosis of SGC-7901 cells by staining with annexinV-FITC/PI. The expression levels of Fas/FasL in SGC-7901 cells were monitored by FCM analysis using an indirect immunofluorescence method. Activity of caspase-3 enzyme was measured by spectrofluorometry. RESULTS: MTT assay showed that matrine inhibited SGC-7901 cells proliferation in a dose-dependent and time-dependent manner. Apoptosis induction was demonstrated by morphological changes under electron microscope and FCM analysis. Fluorescence intensity levels of Fas and FasL were found to be equally up-regulated after matrine treatment, which were both correlated with apoptosis rate. The activity of caspase-3 enzyme increased in matrine groups, positively correlated with apoptosis rate. CONCLUSIONS: Matrine could inhibit cell proliferation and induce apoptosis of SGC-7901 cells in vitro. The apoptosis induction appears to proceed by up-regulating Fas/FasL expression and activating caspase-3 enzyme.

Scutellaria barbate extract induces apoptosis of hepatoma H22 cells via the mitochondrial pathway involving caspase-3.

AIM: To study the growth inhibitory and apoptotic effects of Scutellaria barbata D.Don (S. barbata) and to determine the underlying mechanism of its antitumor activity in mouse liver cancer cell line H22. METHODS: Proliferation of H22 cells was examined by MTT assay. Cellular morphology of PC-2 cells was observed under fluorescence microscope and transmission electron microscope (EM). Mitochondrial transmembrane potential was determined under laser scanning confocal microscope (LSCM) with rhodamine 123 staining. Flow cytometry was performed to analyze the cell cycle of H22 cells with propidium iodide staining. Protein level of cytochrome C and caspase-3 was measured by semi-quantitive RT-PCR and Western blot analysis. Activity of caspase-3 enzyme was measured by spectrofluorometry. RESULTS: MTT assay showed that extracts from S. barbata (ESB) could inhibit the proliferation of H22 cells in a time-dependent manner. Among the various phases of cell cycle, the percentage of cells in S phase was significantly decreased, while the percentage of cells in G(1) phase was increased. Flow cytometry assay also showed that ESB had a positive effect on apoptosis. Typical apoptotic morphologies such as condensation and fragmentation of nuclei and blebbing membrane of apoptotic cells could be observed under transmission electron microscope and fluorescence microscope. To further investige the molecular mechanism behind ESB-induced apoptosis, ESB-treated cells rapidly lost their mitochondrial transmembrane potential, released mitochondrial cytochrome C into cytosol, and induced caspase-3 activity in a dose-dependent manner. CONCLUSION: ESB can effectively inhibit the proliferation and induce apoptosis of H22 cells involving loss of mitochondrial transmembrane potential, release of cytochrome C, and activation of caspase-3.

[Antitumor and immune-modulating effects of Scutellaria barbata extract in mice bearing hepatocarcinoma H22 cells-derived tumor].

OBJECTIVE: To investigate the effects of Scutellaria barbata extract (ESB) in suppressing tumor growth and modulating the immune functions in mice bearing tumors derived from hepatocarcinoma H22 cells. METHODS: Fifty mice inoculated subcutaneously with H22 cells were equally divided into the model group, high-, moderate-, and low-dose ESB groups, and 5-Fu group, with corresponding treatments for 10 days. Another 10 mice with only saline injection served as the normal control group. The body weight, tumor mass, thymus index and spleen index of the mice were measured, and the lymphocyte proliferation activity, NK cell activity and interleukin-2 (IL-2) production by the splenocytes were detected. RESULTS: Moderate- and high-dose ESB significantly suppressed the tumor growth with tumor inhibition rate of 28.68% and 36.98%, respectively. ESB treatment at moderate and high doses significantly increased the thymus index and spleen index (P < 0.01), which were decreased significantly in 5-Fu group. The lymphocyte proliferation activity, NK cell activity and IL-2 production by the splenocytes were significantly lower in the model group than in the normal group (P < 0.05). Compared with the model group, ESB at the high dose obviously increased the three indexes above mentioned. The NK cell activity was also significantly improved in moderate-dose ESB group (P < 0.05). CONCLUSION: ESB can suppress the growth of H22 implant tumor and enhance the immune function of the tumor-bearing mice.

[Effects of alprostadil in prevention of portal vein thrombogenesis after splenectomy and devascularization: a clinical observation of 76 patients with portal hypertension].

OBJECTIVE: To investigate the effects of alprostadil (Lipo PGE1) in prevention of portal vein thrombogenesis (PVT) after splenectomy for portal hypertension. METHODS: Seventy-six patients with portal hypertension undergoing splenectomy and pericardial devascularization were randomly divided into 2 groups :treatment group (n = 40), receiving intravenous drip of injection of radix Salviae miliorrhazae (RSM) 40 ml and alprostadil 20 microg, both once a day since the third day after operation for 2 weeks and then oral administration of dropping pill of SM, and control group (n = 36), receiving intravenous drip of injection of RSM and taking enteric coated aspirin 3 times a day for 2 weeks and then taking dropping pill of SM. Platelets (PLT), prothrombin time (PT), and liver function were detected periodically. Color Doppler ultrasonography was conducted every week to observe the blood flow velocity and diameter of the portal and splenic veins, and if PVT event and ascites occurred. All patients were followed up for 8 - 20 months. RESULTS: No prolongation of coagulation time and bleeding tendency was found in both groups. The PLT number increased remarkably in the 7th to 14th days after operation without significant difference between the 2 groups (P >0.05). The PVT rate of the treatment group was 5.0%, significantly lower than that of the control group (25.0%, chi2 = 6.12, P < 0.05). The ascites rate of the treatment group was 10.0%, significantly lower than that of the control group (33.3%, chi2 = 7.44, P <0.01). The levels of ALT and total bilirubin 7 and 16 days after operation of the treatment group were all significantly lower than those of the control group (all P <0.05). CONCLUSION: Use of alprostadil early after devascularization is an effective and safe measure to prevent PVT, improve liver function, and decrease ascites rate.

[Scutellaria barbata extract enhances efficacy and reduces toxicity of chemotherapy in hepatoma H22-bearing mice].

OBJECTIVE: To study the assistant effect of Scutellaria barbata extract (ESB) in 5-fluorouracil (5-FU) chemotherapy. METHODS: A mouse model of transplanted hepatoma H22 was used in this study to evaluate the synergic and attenuating effects of ESB in chemotherapy. Tumor inhibition rate, life span of mice and the toxicity of chemotherapy were observed. The body weight, tumor weight, thymus index and spleen index in H22-bearing mice were also measured. The phagocytotic function of macrophages was studied by observing phagocytization of peritoneal macrophages. RESULTS: The increase of body weight in 5-FU plus ESB groups was higher than that in 5-FU group, and the side effects such as anorexia, abdominal distention and athrepsy were relieved. Compared with untreated group, prolonged lifetime in 5-FU plus high-dose ESB group and 5-FU plus low-dose ESB group was improved. Life prolongation rates in 5-FU plus high-dose ESB group and 5-FU plus low-dose ESB group were 61.46% and 23.59% respectively. High-dose ESB, 5-FU, 5-FU plus low-dose ESB and 5-FU plus high-dose ESB could inhibit the tumor growth, and the tumor inhibition rates were 36.98%, 42.26%, 52.45% and 65.28%, respectively. Thymus index and spleen index were increased significantly in 5-FU plus low-dose ESB group and 5-FU plus high-dose ESB group. White blood cell (WBC) count was decreased obviously in 5-FU group, while the count of WBC was increased in 5-FU plus low-dose ESB group and 5-FU plus high-dose ESB group. The phagocytotic function of macrophages was also increased in 5-FU plus low-dose ESB group and 5-FU plus high-dose ESB group. CONCLUSION: ESB can enhance the effect of chemotherapy, relieve the side effects and improve immune function of mice in chemotherapy. These results suggest that ESB, as a biochemical modulator to enhance the therapeutic effects, is useful in cancer chemotherapy.

[Effects of Scutellaria Barbata drug-containing serum on apoptosis and mitochondrial transmembrane potential of hepatoma H22 cells].

OBJECTIVE: To investigate the effects of serum containing Scutellaria Barbata extract (ESB) on apoptosis rate and mitochondrial transmembrane potential (MTP) of liver cancer cell line H22 from mice in vitro. METHODS: H22 cells were cultured in vitro and divided into 5 groups: blank control group, low-dose ESB group, medium-dose ESB group, high-dose ESB group and fluorouracil (5-Fu) group. Methyl thiazolyl tetrazolium assay was utilized to determine the proliferation rates of H22 cells. Cellular morphology was observed under a transmission electron microscope (EM). The rhodamine 123 was used as a fluorescence probe to label the H22 cells, and the fluorescence intensities were observed with a laser scanning confocal microscope. The fluorescence intensity of H22 cells indicated the MTP of H22 cells. RESULTS: The inhibition of serum containing ESB on the proliferation of H22 cells in vitro was observed in a time-dependent manner. The typical morphological changes of apoptosis were observed after incubation with ESB-containing serum in high dose for 48h. The apoptosis rates of blank control group, 5-Fu group, low-dose ESB group, medium-dose ESB group and high-dose ESB group were (0.51+/-0.32)%, (11.26+/-2.97)%, (1.07+/-0.46)%, (3.15+/-1.12)%, (7.83+/-2.25)% respectively. ESB could reduce the MTP of H22 cells from mice as compared with the untreated group. The MTPs of the blank control group, 5-Fu group, and low-, medium- and high-dose ESB groups were (245.45+/-67.37), (127.42+/-41.35), (213.68+/-65.52), (186.34+/-56.37) and (142.65+/-39.44) respectively, which were negatively correlated with the apoptosis rates. CONCLUSION: ESB-containing serum effectively induces apoptosis, which may be related to the decrease of MTP in H22 cells.

[Anti-proliferative and apoptosis-inducing activity of Scutellaria barbate containing serum on mouse's hepatoma H22 cells].

OBJECTIVE: To investigate the effects of Scutellaria barbate extract (ESB) on suppressing proliferation and inducing apoptosis of mouse hepatoma H22 cells. METHODS: H22 cells cultured in vitro were divided into 5 groups: blank control group, ESB in high, medium, low dose groups and 5-Fu group. H22 cells were cultured in media with serum containing different concentrations of ESB and blank serum. The proliferation of H22 cells was determined by microculture tetrazolium (MTT) assay. Fluorescence microscopy was utilized to observe the apoptosis of H22 cells by staining with Hoechst 33258. The cell cycle and apoptosis were analyzed by flow cytometry (FCM). RESULTS: The inhibition of serum containing ESB on the proliferation of H22 cells in vitro was observerd in a dose and time dependent manner. The typical morphological changes of apoptosis were observed after incubation with ESB-containing serum in high dose for 48 hours. Among the various phases of cell cycle, the percentage of cells in S phase decreased significantly, while the percentage of cells in G1 phase increased. Drug-containing serum showed positive effect on cell apoptosis. The apoptosis rate of blank control group, ESB in low, medium, high dose groups and 5-Fu group were 0.51%, 1.07%, 3.15%, 7.83%, 11.26%, respectively. CONCLUSION: ESB containing serum can inhibit proliferation and induce apoptosis of H22 cells in vitro.

[Apoptotic mechanism of gastric carcinoma cells induced by matrine injection].

OBJECTIVE: To investigate the mechanism of gastric carcinoma cells apoptosis induced by matrine injection in vitro. METHODS: Effects of 24, 48, 72, 96 h incubation with different concentrations (0.25-1.5 g/L) of matrine injection on proliferation of SGC-7901 cells were evaluated using 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay. The cellular morphology of SGC-7901 cells was observed by transmission electron microscope (EM). Flow cytometry was used to analyze the apoptosis of SGC-7901 cells by staining with annexin V-FITC/PI. The expression of Fas/FasL was examined by flow cytometry using specific antibody. The activity of caspase-3 was measured by spectrofluorometry. RESULTS: Matrine injection could inhibit the proliferation of SGC-7901 cells in a dose- and time-dependent manner. The typical morphological changes of apoptosis were observed after incubation with 1.0 g/L matrine injections for 48 h. The apoptosis rates of 0.5 g/L, 1.0 g/L and 1.5 g/L groups were 39.80%, 58.11% and 79.00% respectively. The apoptotic cells in matrine injection group were mainly early apoptotic cells, and those in 5-FU group were mainly late apoptotic cells and necrotic cells. Spectrofluorometry revealed FI levels of Fas and FasL were equal, which were both correlated with apoptosis rate. The activity of caspase-3 increased with the elevation of matrine concentration, and was correlated with the apoptosis rate. CONCLUSION: Matrine injection can induce apoptosis of SGC-7901 cells through the up-regulation of Fas/FasL expression and activation of caspase-3.

[Therapeutic effects of a recombinant adeno-associated virus expressing fusion peptide NT4p53(C22)Ant on ICR mice bearing transplanted liver cancer].

OBJECTIVE: To study the therapeutic effects of recombinant adeno-associated virus (rAAV) expressing fusion peptide NT4p53(C22)Ant against transplanted liver cancer in ICR mice. METHODS: NT4p53(C22)Ant was constructed, subcloned into recombinant AAV vector, and amplified in 293 packaging cells. The efficacy of rAAV-NT4p53(C22)Ant on tumors derived from H22 cells inoculated subcutaneously in IRC mice was evaluated according to the tumor weight, inhibition rate, survival time of the mice and the histological findings. RESULTS: A single dose of rAAV-NT4p53(C22)Ant of 100 microl (2 x 10(11) pfu/ml) injected into the transplanted H22 tumors in the ICR mice resulted in tumor disappearance in 7 (totally 12) mice, and death occurred in only 1 mouse. The injection also resulted in decreased tumor weight and prolonged survival of the mice (for over 70 days). All the 7 mice with only rAAV injection or no treatment all died, with a mean survival of about 30 days. The tumor inhibition rate exceeded 90% in mice with rAAV-NT4p53(C22)Ant injection, significantly higher than that of mice without the injection. The histological examination revealed significantly decreased tumor cells in mice with rAAV-NT4p53(C22)Ant injection as compared with those without such treatment. CONCLUSION: rAAV-NT4p53(C22)Ant can induce apoptosis of the H22 tumor cells transplanted in IRC mice to inhibit the tumor growth and prolong the survival of the mice.

Infusion of donor hepatic non-parenchymal cells prolongs survival of skin allografts in mice: role of microchimerism and IL-4.

BACKGROUND: In the mouse skin allograft model, specific immune tolerance to the donor was induced by injection of donor hepatic non-parenchymal cells (NPCs). This markedly prolonged the survival time of the allograft. The mechanism of the induction of immune tolerance with donor hepatic NPCs is thought to be related to microchimerism and the IL-4 level. This work aimed at exploring the way of inducing immune tolerance and understanding the mechanism. METHODS: C57BL/6 (B6) mice were primed by intravenous injection of 2 x 10(7) NPCs from C3H mice. Cyclophosphamide (200 mg/kg) was injected intraperitoneally 48 hours later. Eighteen days after the NPC injection, skin from C3H mice was transplanted to B6 mice and the survival of the grafts was assessed. The immune reaction of splenocytes from the treated B6 mice to donor-specific T-cells was measured by 3H-TdR incorporation. Microchimerism in the spleen was determined by flow cytometric analysis sytem (FCAS) analysis, and the serum level of IL-4 was assayed by ELISA at designed times. RESULTS: The survival time of the skin graft was markedly prolonged from 10 days to 70 days in controls. Microchimerism in the spleen was found as early as day 1 post-NPC injection, then it increased steadily, and there was a positive relationship between graft survival and the quantity of microchimerism. The ELISA results showed that NPC infusion enhanced IL-4 production, especially in the mice with longer graft survival. CONCLUSION: Donor NPC infusion pre-transplant can prolong the survival of the skin graft and microchimerism and high levels of IL-4 may be involved.

[The study of the effect of antisense oligonucleotide specific to K-ras point mutation on human pancreatic carcinoma cell PC-2].

OBJECTIVE: To investigate the effects of antisense oligonucleotide specific to K-ras point mutation on human pancreatic carcinoma cell PC-2 in vitro. METHODS: Human pancreatic carcinoma cell PC-2 was transducted with antisense oligonucleotide specific to K-ras point mutation by liposome; the expression of target gene was studied with immunohistochemistry and in situ hybridization. The effect on cell proliferation was studied by artificial count, MTT and mass test. RESULTS: The expression degree of ras protein and K-ras mRNA transducted with antisense oligonucleotide decreased apparently compared with control group and sense oligonucleotide group 48 h after tansduction. The inhibitory effect on cell proliferation was confirmed by artificial count, MTT and mass test. CONCLUSIONS: Antisense oligonucleotide specific to K-ras point mutation has an apparent inhibitory effect on target gene expression and cell proliferation of human pancreatic carcinoma cell in vitro.