Knockdown of PYCR1 inhibits proliferation, drug resistance and EMT in colorectal cancer cells by regulating STAT3-Mediated p38 MAPK and NF-κB signalling pathway.

PYCR1 exerts an important role in various cancers, but its effect on colorectal cancer (CRC) and the potential mechanism remain to be clarified. In this study, we aimed to explore the effect of PYCR1 on CRC and further explore the special molecular mechanism. The expression of PYCR1 in CRC tissues and cells was analysed by RT-PCR assay. Cell proliferation was explored using an MTT assay. A CoIP assay was performed to determine the binding activity of PYCR1 and STAT3. Western blot was used to measure the protein expression of P-gp, MRP1, E-cadherin and vimentin. The results revealed that PYCR1 is highly expressed in CRC tissues and cells. PYCR1-siRNA inhibited the proliferation, drug resistance and epithelial-mesenchymal transition (EMT) of CRC cells. The CoIP assay result demonstrated that PYCR1 interacts directly with STAT3, and STAT3 overexpression partly reverses the effect of PYCR1 on proliferation, drug resistance and EMT of CRC cells. What is more, si-PYCR1 inhibited STAT3-mediated p38 MAPK and NF-κB signalling pathways. Collectively, it suggests that knockdown of PYCR1 inhibits proliferation, drug resistance and EMT potentially by regulating STAT3-mediated p38 MAPK and NF-κB signalling pathways in CRC cells.

ABCA1 inhibits PDGF-induced proliferation and migration of rat airway smooth muscle cell through blocking TLR2/NF-κB/NFATc1 signaling.

Airway remodeling is a key feature of asthma, characterized by abnormal proliferation and migration of airway smooth muscle cells (ASMCs). ABCA1, a member of the ATP-binding cassette family of active transporters, plays an essential role in the progression of lung diseases. However, the contributions of ABCA1 in ASMCs remain to be explored. The purpose of the present study was to investigate the functional role and potential molecular mechanism of ABCA1 in platelet derived growth factor (PDGF)-induced primary rat ASMC proliferation and migration. We observed that PDGF- led to a significant decrease in the expression of ABCA1. Overexpression of ABCA1 strikingly suppressed PDGF-induced ASMC proliferation accompanied by a decrease in the expression of PCAN stimulated by PDGF. Additionally, augmentation of ABCA1 dramatically restrained PDGF-induced migration concomitant with attenuate the accumulation of MMP-2 and MMP-9 in response to PDGF. Furthermore, forced expression of ABCA1 enhanced contractile phenotype markers proteins including α-SMA along with sm-MHC, sm-α-actin, and calponin reduced by PDGF. Meanwhile, introduction of ABCA1 depressed ECM over-deposition induced by PDGF as reflected by a decrease in the expression of ECM protein collagen I and fibronectin. More importantly, addition of ABCA1 effectively suppressed the activity of TLR2/NF-κB signaling as well as diminished the expression of NFATc1 in rat ASMCs after PDGF stimulation. Interestingly, blockage of TLR2/NF-κB signaling effectively inhibited PDGF-induced proliferation and migration, these effects were similar to ABCA1. Taken together, these data implicated that ABCA1 suppressed PDGF-induced proliferation, migration, and contraction in rat ASMCs at least partly through TLR2/NF-κB/NFATc1 signaling, which might offer hope for the future treatment of airway remodeling in asthma.

Combining Sprague-Dawley rat uterus cell membrane chromatography with HPLC/MS to screen active components from Leonurus artemisia.

CONTEXT: Leonurus artemisia (Lour.) S.Y.Hu (Lamiaceae) (YiMuCao in Chinese) is a traditional Chinese medicine. Leonurus artemisia has been shown to have many pharmacological effects such as increasing uterine contraction amplitude, and tension, but the active components are still unknown. OBJECTIVE: The objective of this study is to determine active components of L. Artemisia that are responsible for the biological activity using HPLC and cell membrane-based system. MATERIALS AND METHODS: The whole L. artemisia ethanol extract and its eight fractions were screened using Sprague-Dawley rat uterus cell membrane chromatography (CMC) combined with the HPLC/MS system. Oxytocin was used to investigate the activity of CMC column. The effect of active components screened from L. artemisia was studied by tension measurement of isolated rat uterine strips in vitro at a dose of 10(-7)-10(-4 )mol/L with oxytocin as a control. RESULTS: The acetone extract showed obvious activity when compared with the eight extracts of L. artemisia. From the acetone extract, in the negative ionization mode, the active compound was identified as genkwanin, with a molecular weight of 283. In vitro pharmacological experiments proved that genkwanin promoted uterine contractions at a dose from 10(-7) to 10(-4 )mol/L. The EC50 value was 4.86 ± 4.21 µmol/L for genkwanin and 4.30 ± 3.65 µmol/L for oxytocin on the contractile amplitude of uterine strips isolated from rats. DISCUSSION AND CONCLUSION: Genkwanin was identified as the active compound in L. artemisia by this method. In vitro pharmacological experiments proved that genkwanin promoted uterine contractions. Genkwanin may be used to uterine inertia and may have an effect on postpartum hemorrhage.

Role of heparin on serum VEGF levels and local VEGF contents in reducing the severity of experimental severe acute pancreatitis in rats.

OBJECTIVE: The aims of this study were to examine the effects of prophylactic heparin treatment during taurocholate-induced pancreatitis in rats and its impact on serum VEGF levels and local VEGF contents within the pancreas. METHODS: Severe acute pancreatitis (SAP) was induced by injecting 4% sodium taurocholate into the pancreatic duct. Heparin at a dose of 150 IU/kg s.c. was administered 30 min before the operation. The rats were sacrificed 1 h, 3 h, 6 h and 12 h (n = 5 per time point) after the onset of pancreatitis. The severity of pancreatitis, serum VEGF levels and local VEGF contents were evaluated with and without heparin pretreatment. RESULTS: The serum VEGF levels increased at an early phase of pancreatitis, and the highest level was found at 12 h after inducing pancreatitis. The gray value of the local VEGF showed a remarkable increase from the onset of the pancreatitis. However, the gray value of VEGF did not show an increase over time but maintained a high level during the entire process. Prophylactic heparin treatment significantly improved the morphologic changes, myeloperoxidase (MPO), TNF-α and malondialdehyde (MDA) activities. Meanwhile, it decreased the serum VEGF levels and the contents of VEGF within the pancreatic tissue. CONCLUSIONS: The present study suggests that prophylactic heparin ameliorates the severity of taurocholate-induced pancreatitis via its anti-inflammatory properties. These protective effects may be partly due to decreasing serum VEGF levels and VEGF contents within the pancreas.

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.

Circulating RNAs, circ_4911 and circ_4302, are novel regulators of endothelial cell function under a hepatocellular carcinoma microenvironment.

Hepatocellular carcinoma (HCC) is a common hypervascular tumor disease. Endothelial cells, as a crucial component of the tumor microenvironment, have been reported to participate in angiogenesis and influence the development of tumors, including HCC. Recent studies have demonstrated that circulating RNAs (circRNAs) participate in the functional regulation of endothelial cells. However, the expression and function of circRNAs in endothelial cells under the HCC microenvironment is still unclear. In the present study, we analyzed the expression profiles and investigated the role of circRNAs in human umbilical vein endothelial cells (HUVECs) co­cultured with human primary hepatoma cells. Based on an RNA­sequencing assay, we screened 19 significantly downregulated circRNAs in HUVECs under an HCC microenvironment. Subsequently, we validated the expression of the candidate circRNAs using RT­qPCR, and selected two of the most downregulated circRNAs among them, circ_4911 and circ_4302. Next, through circRNA overexpression experiments, we demonstrated that overexpression of circ_4911 and circ_4302 both inhibited the proliferation and migration of HUVECs, and arrested cells at the GO/G1 stage, while promoting adhesion. Overall, in the present study, we identified the roles of circ_4911 and circ_4302 in regulating functions of HUVECs under an HCC microenvironment.

[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.

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.

[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.

The effect-enhancing and toxicity-reducing action of the extract of herba Scutellariae barbatae for chemotherapy in hepatoma H22 tumor-bearing mice.

OBJECTIVE: To investigate the effect-enchancing and toxicity-reducing action of the extract of Ban Zhi Lian (Herba Scutellariae Barbatae, EHSB) for chemotherapy in hepatoma H22 tumor-bearing mice. METHODS: The tumor-bearing mice were divided into 6 groups randomly: a model group, a high dose EHSB group, a low dose EHSB group, a 5-fluorouracil (5-FU) group, a 5-FU+high dose EHSB group and a 5-FU+low dose EHSB group, and with a normal group set as the controls. All the groups were treated for 10 days. The life prolongation rate, toxic reactions of chemotherapy, WBC count, the body weight, tumor weight, thymus index and spleen index, and phagocytic function of intra-abdominal macrophages were investigated in the H22 tumor-bearing mice. RESULTS: The increase of the body weight in both the 5-FU+EHSB groups was significantly higher than that in the 5-FU group, with the toxic reactions such as anorexia, abdominal distension and emaciation significantly alleviated. Growth of the tumor was significantly inhibited in the high dose EHSB group, the 5-FU group, the 5-FU+high dose EHSB group, and the 5-FU+low dose EHSB group. The survival time in the 5-FU+high dose EHSB group and the 5-FU+low dose EHSB group was significantly prolonged as compared with that of the 5-FU group. The life prolongation rate was 98.72% in the 5-FU+high dose EHSB group and 52.11% in the 5-FU+low dose EHSB group. Growth of the transplanted tumor was significantly inhibited in the high dose EHSB group, the 5-FU group, the 5-FU+high dose EHSB group, the 5-FU+low dose EHSB group. The tumor inhibition rate in the high dose EHSB group, the 5-FU group, the 5-FU+high dose EHSB group and the 5-FU+low dose EHSB group was 36.98%, 42.26%, 65.28% and 52.45%, respectively. 5-FU combined with a high-dose EHSB could significantly enhance the tumor inhibition rate (P < 0.05). The thymus index and the spleen index significantly increased in the high dose EHSB group, and atrophy of the immunological organs induced by chemotherapy was improved in the 5-FU+high dose EHSB group and in the 5-FU+low dose EHSB group. The WBC count decreased significantly in the 5-FU group, but increased in both the 5-FU+EHSB groups. The phagocytic function of intra-abdominal macrophages was increased in both the 5-FU+EHSB groups, with the phagocytic rate and the phagocytic index increased by 78.55% and 81.63% in the 5-FU+high dose EHSB group and by 43.97% and 44.90% in the 5-FU+low dose EHSB group. CONCLUSIONS: EHSB can significantly enhance the tumor inhibition rate of 5-FU, reduce the toxic effects, prolong the survival time, and improve immune function in the H22 tumor-bearing mice.

Clinical effects of shenqi fuzheng injection in the neoadjuvant chemotherapy for local advanced breast cancer and the effects on T-lymphocyte subsets.

OBJECTIVE: To evaluate clinical effects of shenqi fuzheng Injection ([Chinese characters: see text]) in the neoadjuvant chemotherapy for local advanced breast cancer and the effects on T-lymphocyte subsets. METHODS: During the period from 2000 to 2005, 126 patients with local advanced breast cancer were treated with the neoadjuvant chemotherapy. They were randomly divided into the following two groups: a control group of 61 cases treated by chemotherapy alone and a study group of 65 cases treated by chemotherapy plus shenqi fuzheng injection. All the cases of both groups were given the CEF (CTX 500 mg/m2, d1,8; EPI 40 mg/m2, d1, 8; and 5-Fu 500 mg/m2 d1,8) regimen. The clinical effects, the effects on T-lymphocyte subgroup and NK cells, and the toxic side effects were observed. RESULTS: All the patients completed two cycles of the chemotherapy, and the efficacy and the toxic side effects were evaluated. For the primary tumor in the breast, the total effective rate was 69.2% (45/65) in the study group and 49.2% (30/61) in the control group with a statistically significant difference in the intergroup comparison (chi2 = 5.251, P = 0.022, < 0.05). There was no progression of the disease in both the groups, and there were no grade IV toxic side effects in the two groups. The major toxic responses were myelosuppression and gastrointestinal reaction, which were milder in the study group than the control group, and with a shorter recovery course in the former than the latter. Besides, an obvious rise of the T-lymphocyte subgroup and NK cells was found in the study group after the neoadjuvant chemotherapy, with a very significant difference from the controls (P < 0.01). CONCLUSIONS: Shenqi fuzheng Injection can improve and regulate immune function of the patients with local advanced breast cancer given the neoadjuvant chemotherapy, and therefore it can enhance the curative effect and reduce the side effect as well.

[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.

[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.

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.

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.

Value of ultrasound in the diagnosis of congenital renal malformations.

OBJECTIVE: To assess the value of ultrasound in diagnosis of congenital renal malformations. METHODS: The records of obstetric ultrasound examinations were reviewed in all the pregnant women admitted in Alta Bates Perinatal Diagnostic center (Oakland, California, USA) during 5 years. RESULTS: Ultrasound examination identified 58 cases of congenital renal malformations of different kinds, including 4 cases of renal agenesis, 8 multicystic dysplastic kidney, 5 cystic renal dysplasia with obstructions, 6 renal and ureteral duplications, 6 ectopic kidneys, 18 hydronephroses, 3 autosomal recessive polycystic kidney diseases, 2 autosomal dominant polycystic diseases, 1 Finnish-type congenital nephrosis, 3 Meckel-Gruber syndromes, and 2 Beckwith-Wiedemann syndromes. Different renal malformations had different ultrasound findings that correlated to abnormalities in embryonic developments. CONCLUSIONS: Urinary tract abnormalities have a profound effect on pregnancy outcome, especially when associated with oligohydramnios. Many urinary anomalies can be readily detected and diagnosed by ultrasound, which provides a useful modality in diagnosis of fetal congenital renal malformations. Clear understanding of the causes of these abnormalities facilitates prognostic evaluation and clinical decision on the treatment protocol.

[Effects of Ras antisense oligoribonucleotide on multidrug resistance of pancreatic carcinoma Pc-2 cells].

OBJECTIVE: To investigate the role of Ras antisense oligoribonucleotide (ASODN) in multidrug resistance (MDR) of pancreatic carcinoma Pc-2 cells. METHODS: Ras and P-gp expression was suppressed by Ras ASODN. Sensitivity of Pc-2 cells to chemotherapy was determined by the MTT assay. MDR-1 mRNA level was detected by fluorogenic probe quantitative reverse transcription polymerase chain (RT-PCR) method. Flow cytometry (FCM) was used to detect the accumulative concentration of adriamycin (ADR) in the cells. RESULTS: Ras ASODN significantly inhibited the Ras and P-gp expression (P < 0.05), increased the sensitivity of Pc-2 cells to chemotherapeutic agents (P < 0.05), decreased MDR-1 gene level in Pc-2 cells (P < 0.05), and increased the intracellular intake of ADR in Pc-2 cells (P < 0.05). CONCLUSION: Ras ASODN may enhance the sensitivity of multidrug-resistant pancreatic cancer Pc-2 cells to chemotherapeutic agents by regulating MDR-1 gene level.

[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.

Polymerase synthesis and potential interference of a small-interfering RNA targeting hPim-2.

AIM: To synthesize three small-interference RNAs (siRNAs) by T7 RNA polymerase-catalyzed reaction, and to investigate their efficacy on modulating the expression of serine/threonine kinase Pim-2 in human colon cancer cell line. METHODS: siRNA I, II and III were synthesized by T7 RNA polymerase-directed in vitro transcription, then transfected into human colon cancer cells SW-480. After incubation for 6 h at 37 degrees, 100 mL/L FBS in RPMI 1640 was substituted in each well. After the transfection was repeated twice to three times in each kind of siRNA, hPim-2 mRNA and protein expression were measured by RT-PCR and Western blotting, respectively. RESULTS: Compared to the control group, after transfected for 48 h with hPim-2 siRNA I, II and III, the relative inhibition rates of hPim-2 mRNA expression in colon cancer cells were 65.4% (P<0.05), 46.2% (P<0.05) and 56.1% (P<0.05), respectively. The protein level of hPim-2 was decreased at 72 h compared to the untransfected cells. The relative inhibition percentages of hPim-2 protein by siRNA I, II, III were 61.6% (P<0.05), 45.8% (P<0.05) and 55.6% (P<0.05), respectively. CONCLUSION: The in vitro transcribed siRNAs can be useful for silencing oncogene hPim-2 expression specifically and efficiently. This may open a new path toward the use of siRNAs as a gene-specific therapeutic tool.