多囊脾治验案

多囊脾治验案郑祖纯向某，女，４０岁，１９９２年７月，始发腹胀，腹痛，左下肋边缘疼痛，仰卧时可触及到鸡蛋大一包块．不移动，劳累后加剧，在当地医院治疗半年后无效，且包块逐渐增大。１９９３年２月．Ｂ超捡查；肝、胆、胰显示清楚，未见异常回声，脾脏明显增大，肋...

Down-regulation of extracellular signal-regulated kinase 1/2 activity in P-glycoprotein-mediated multidrug resistant hepatocellular carcinoma cells

AIM： To study the expression and phosphorylation of extracellular signal-regulated kinase （ERK） i and ERK2 in multidrug resistant （MDR） hepatocellular carcinoma （HCC） cells.METHODS： MDR HCC cell lines, HepG2/adriamycin （ADM） and SMMC7721/ADM, were developed by exposing parental cells to stepwise increasing concentrations of ADM. MTT assay was used to determine drug sensitivity. Flow cytometry was employed to analyze cell cycle distribution and measure cell P-glycoprotein （P-gp） and multidrug resistant protein 1 （MRP1） expression levels. ERK1 and ERK2 mRNA expression lev-ls were measured by quantitative real-time PCR （QRTPCR）. Expression and phosphorylation of ERK1 and ERK2 were analyzed by Western blot.RESULTS： MTT assay showed that HepG2/ADM andSMMC7721/ADM were resistant not only to ADM, but also to multiple anticancer drugs. The P-gp expression was over 10-fold higher in HepG2/ADM cells than in HepG2 cells （8.92% ±0.22% vs 0.88% ± 0.05%, P 〈 0.001） and over 4-fold higher in SMMC7721/ADM cells than in SMMC7721 cells （7.37% ± 0.26% vs 1.74% ± 0.25%, P 〈 0.001）. However, the MRP1 expression was not significantly higher in HepG2/ADM and SMMC7721/ADM cells than in parental cells. In addition, the percentage of MDR HepG2/ADM and SMMC7721/ADM cells was significantly decreased in the G0/G1 phase and increased in the the S phase or G2/M phase. QRT-PCR analysis demonstrated that the ERK1 and ERK2 mRNA expression increased apparently in HepG2/ADM cells and decreased significantly in SMMC7721/ADM cells. Compared with the expression of parental cells, ERK1 and ERK2 protein expressions were markedly decreased in SMMC7721/ADM cells. However, ERK2 protein expression was markedly increased while ERK1 protein expression had no significant change in HepG2/ADM cells. Phosphorylation of ERK1 and ERK2 was markedly decreased in both HepG2/ADM and SMMC7721/ADM MDR cells.CONCLUSION： ERK1 and ERK2 activities are downregulated in P-gp-mediated MDR HCC cells. ERK1 or ERK2 might be a potential drug target for circumventing MDR HCC cells,

Effect of recombinant human interleukin-11 on expressions of interleukin-11 receptorα-chain and glycoprotein 130 in intestinal epithelium cell line-6 after neutron irradiation

AIM： To explore the effect of recombinant human interleukin-11 （rhIL-11） on the expressions of interleukin-11 receptor α-chain （IL-11Rα） and an additional signal transducer glycoprotein 130 （gp130） in intestinal epithelium cell line-6 （IEC-6） after neutron irradiation. METHODS： Cultured IEC-6 cells were exposed to 4.0Gy neutron and treated with 100 ng/mL rhIL-11 12 h prior to or immediately after irradiation. The apoptosis and necrosis rates and expressions of IL-11Rα and gp130 were observed by flow cytometry, immunohistochemistry, Western blot and image analysis. RESULTS： The apoptosis rate of IEC-6 cells was increased by irradiation at 6 h （P 〈 0.01）, IL-11 stimulation resulted in a decreased apoptosis rate in irradiated IEC-6 cells （P 〈 0.05）. In normal control IEC-6 cells, intense immunoreactivity of IL-11Rα was located within the cell membrane and cytoplasm. The level of IL-11Rα expression significantly decreased at 6 h after irradiation （P 〈 0.01） and restored at 24 h after irradiation. In IEC-6 cells treated with both radiation and rhIL-11, the level of IL- 11Rα expression was higher than that of irradiated cells （P 〈 0.05）. When it came to gp130 protein, it was located in the cytoplasm of IEC-6 cells. After irradiation, we found a progressive decrease in the expression of gp130 protein （P 〈 0.05） in 48 hours post-radiation, while in rhIL-11-stimulated cells, it came back to normal level at 24 h after irradiation and decreased at 48 h, but was still higher than that of only irradiated cells （P 〈 0.05）. CONCLUSION： rhIL-11 can protect IEC-6 cells from neutron irradiation. The protective effect of rhIL-11 might be connected with its ability to up-regulate the expressions of specific ligand-binding subunit IL-11Rα and signal-transducing subunit gp130.

Astragalus polysaccharides can regulate cytokine and P-glycoprotein expression in H22 tumor-bearing mice

AIM:To investigate the adjunct anticancer effect of Astragalus polysaccharides in H22 tumor-bearing mice.METHODS:To establish a solid tumor model,5.0 × 10 6 /mL H22 hepatoma cells were inoculated subcutaneously into the right armpit region of Kunming mice(6-12 wk old,18-22 g).When the tumors reached a size of 100 mm 3,the animals were treated as indicated,and the mice were randomly assigned to seven groups(n = 10 each).After ten days of treatment,blood samples were collected from mouse eyes,and serum was harvested by centrifugation.Mice were sacrificed,and the whole body,tumor,spleen and thymus were weighed immediately.The rate of tumor inhibition and organ indexes were calculated.The expression levels of serum cytokines,P-glycoprotein(P-GP) and multidrug resistance(MDR) 1 mRNA in tumor tissues were detected using enzyme-linked immunosorbent assay,Western blotting,and quantitative myeloid-derived suppressor cells reverse transcription-polymerase chain reaction,respectively.RESULTS:The tumor inhibition rates in the treatment groups of Adriamycin(ADM) + Astragalus polysaccharides(APS)(50 mg/kg),ADM + APS(100 mg/kg),and ADM + APS(200 mg/kg) were significantly higher than in the ADM group(72.88% vs 60.36%,P = 0.013;73.40% vs 60.36%,P = 0.010;77.57% vs 60.36%,P = 0.001).The spleen indexes of the above groups were also significantly higher than in the ADM group(0.65 ± 0.22 vs 0.39 ± 0.17,P = 0.023;0.62 ± 0.34 vs 0.39 ± 0.17,P = 0.022;0.67 ± 0.20 vs 0.39 ± 0.17,P = 0.012),and the thymus indexes of the ADM + APS(100 mg/kg) and ADM + APS(200 mg/kg) groups were significantly higher than in the ADM group(0.20 ± 0.06 vs 0.13 ± 0.04,P = 0.029;0.47 ± 0.12 vs 0.13 ± 0.04,P = 0.000).APS was found to exert a synergistic antitumor effect with ADM and to alleviate the decrease in the sizes of the spleen and thymus induced by AMD.The expression of interleukin-1α(IL-1α),IL-2,IL-6,and tumor necrosis factor-α(TNF-α) was significantly higher in the ADM + APS(50 mg/kg),ADM + APS(100 mg/kg) and ADM + APS(200 mg/kg) groups than in the ADM group;and IL-10 was significantly lower in the above groups than in the ADM group.APS could increase IL-1α,IL-2,IL-6,and TNF-α expression and decrease IL-10 levels.Compared with the ADM group,APS treatment at a dose of 50-200 mg/kg could downregulate MDR1 mRNA expression in a dose-dependent manner(0.48 ± 0.13 vs 4.26 ± 1.51,P = 0.000;0.36 ± 0.03 vs 4.26 ± 1.51,P = 0.000;0.21 ± 0.04 vs 4.26 ± 1.51,P = 0.000).The expression level of P-GP was significantly lower in the ADM + APS(200 mg/kg) group than in the ADM group(137.35 ± 9.20 mg/kg vs 282.19 ± 20.54 mg/kg,P = 0.023).CONCLUSION:APS exerts a synergistic anti-tumor effect with ADM in H22 tumor-bearing mice.This may be related to its ability to enhance the expression of IL1α,IL-2,IL-6,and TNF-α,decrease IL-10,and downregulate MDR1 mRNA and P-GP expression levels.

The interaction of versican with its binding partners

Versican belongs to the family of the large aggregating chondroitin sulfate proteoglycans located primarily within the extracellular matrix （ECM）. Versican, like other members of its family, has unique N- and C-terminal globular regions, each with multiple motifs. A large glycosaminoglycan-binding region lies between them. This review will begin by outlining these structures, in the context of ECM proteoglycans. The diverse binding partners afforded to versican by virtue of its modular design will then be examined. These include ECM components, such as hyaluronan, type Ⅰ collagen, tenascin-R, fibulin-1, and -2, fibrillin-1, fibronectin, P- and L-selectins, and chemokines. Versican also binds to the cell surface proteins CD44, integrin β1, epidermal growth factor receptor, and P-selectin glycoprotein ligand-1. These multiple interactors play important roles in cell behaviour, and the roles of versican in modulating such processes are discussed.

Action of ADAMs in Animal Growth and Development

The ADAMs are a family of cell surface glycoproteins,which were discovered and identified in recent years.They contain several function domains and play some important roles in the formation of sexual cell,fertilization,embryogenesis,cell fusion,organogenesis,cell differentiation,etc.The action of ADAMs in growth and development is summarized in this paper.

Construction of a fusion protein expression vector MK-EGFP and its subcellular localization in different carcinoma cell lines

AIM： To construct an expression plasmid encoding human wild-type midkine （MK） and enhanced green fluorescence protein （EGFP） fusion protein （MK-EGFP）, and to analyze the subcellular localization of MK in different cardnoma cell lines. METHODS： Two kinds of MK coding sequences with or without signal peptide were cloned into plasmid pEGFP-N2, and the recombinant plasmids constructed were introduced into HepG2, MCF7 and DU145 cells, respectively, by transfection. With the help of laser scanning confocal microscopy, the expression and subcellular localization of MK-GFP fusion protein could be detected. RESULTS： Compared with the GFP control, in which fluorescence was detected diffusely over the entire cell body except in the nucleolus, both kinds of fusion protein MK-GFP were localized exclusively to the nucleus and accumulated in the nucleolus in the three kinds of cancer cell lines. CONCLUSION： This study reveals the specific nucleolar translocation independent of signal peptide, which may be involved in the mechanism that MK works. It provides valuable evidence for further study on the functions of MK in nucleus and its possible mechanisms, in which ribosomal RNA transcription and ribosome assembly are involved.

Effect of berberine on Cdk9 and cyclin T1 expressions in myocardium of diabetic rats

Objective： To investigate the effect of berberine, one of the main alkaloids of Rhizoma coptidis, on myocardial morphology and the expressions of cyclin-dependent kinase 9 （Cdk9） and cyclin T1 protein in the myocardium of type 2 diabetic rats. Methods： Type 2 diabetes mellitus rats were induced by an injection of 35 mg/kg streptozotocin （STZ） and a high-carbohydrate/high-fat diet for 16 weeks. Diabetic rats were given low-, middle-, high-dose berberine （75, 150, 300 mg/kg）, fenofibrate （100 mg/kg） and rosiglitazone （4 mg/kg） for another 16 weeks, respectively. The myocardium structure was observed with hematoxylin ＆ eosin （H＆E） staining and Cdk9 and cyclin T1 protein expressions were detected by immunohistochemistry. Results： Middle-dose, high-dose berberine improved myocardial hypertrophy and interstitial fibrosis of diabetic rats. Cdk9 and cyclin T1 protein were significantly lower in diabetic myocardium than in control one （P〈0.01）, and middle-dose, high-dose berberine and fenofibrate obviously increased both Cdk9 and cyclin T1 expression to near control level （P〈0.01）. Conclusion： Berberine modulates Cdk9 and cyclin T1 protein expression in diabetic myocardium which may contribute to ameliorate myocardium damage.

Relationship between therapeutic efficacy of arterial infusion chemotherapy and expression of P-glycoprotein and p53 protein in advanced hepatocellular carcinoma

AIM： To investigate the relationship between the chemotherapeutic drug efficacy and the expression of P-glycoprotein （PGP） and p53 protein in advanced hepatocellular carcinoma （HCC）. METHODS： The study was conducted on 41 patients with advanced HCC who were treated by repeated arterial infusion chemotherapy. Biopsy specimens from the tumor were collected before the start of treatment in all the patients, and the specimens were stored frozen until immunohistochemical staining, which was performed after the start of treatment, to detect PGP and p53 protein expressions. Twenty of the fortyone patients were treated with an anthracycline drug （epirubicin hydrochloride; anthracycline group）, and the remaining 21 were treated with a non-anthracycline drug （mitoxantrone hydrochloride in 11 patients and carboplatin in 10 patients; non-anthracycline group）. The relationship between the chemotherapeutic efficacy and the results of immunostaining were compared between the two groups. RESULTS： Before the start of the treatment, PGPpositive rate was 90.2% （strongly-positive, 36.6%） and p53 protein-positive rate was 34.1% （strongly-positive, 19.5%）. In the anthracycline group, the response rate was 40.0%. The number of patients showing poor response to the treatment was significantly larger in the patients with strongly positive PGP expression （P= 0.005）, and their prognoses were poor （P= 0.001）. in the nonanthracycline group, the response rate was 42.9%,and there was no significant relationship between the chemotherapeutic drug efficacy and the PGP or p53 protein expression. When only the data from the 11 patients treated with anthraquinone drug, mitoxantrone, were analyzed, however, the number of patients who showed poor response to treatment was significantly higher among the p53-positive patients （P= 0.012）, irrespective of the survival outcome. CONCLUSION： The chemotherapeutic efficacy with an anthracycline drug for advanced HCC can be predicted by immunohistochemical analysis of PGP expression. Similarly, immunostaining to evaluate p53 protein may be useful to predict the response in patients treated with an anthraquinone drug.

Identification of the Interaction between P-Glycoprotein and Anxa2 in Multidrug-resistant Human Breast Cancer Cells

Objective To explore the interaction of Anxa2 with P-Glycoprotein （P-gp） in the migration and invasion of the multidrug-resistant （MDR） human breast cancer cell line MCF-7/ADR. Methods A pair of short hairpin RNA （shRNA） targeting P-gp was transfected into MCF-7/ADR cells, and monoclonal cell strains were screened. The expression of P-gp was detected by Western blot. Transwell chambers were used to observe the cell migration capacity and invasion ability. The interaction between P-gp and Anxa2 was examined by immunoprecipitation and immunofluorescence confocal microscopy analyses. Results P-gp expression was significantly knocked down, and there were notable decreasing trends in the migration and invasion capability of MDR breast cancer cells （P〈0.05）. There was a close interaction between Anxa2 and P-gp. Conclusions MCF-7/ADR is an MDR human breast cancer cell line with high migration and invasion abilities. The knockdown of P-gp notably impaired the migration and invasion abilities of the tumor cells. The interaction of Anxa2 with P-pg may play an important role in time enhanced invasiveness of MDR human breast cancer cells.

P-glycoprotein is not involved in pathway of anti-Fas/Fas-induced apoptosis in KBv200 cells

AIM: To verify whether P-glycoprotein (P-gp) could induce cell resistance to apoptosis by inhibiting caspase-8 and caspase-3.METHODS: Human KB cells, either drug-sensitive or with multidrug resistance (MDR) phenotype caused by overexpression of P-gp (KBv200 cells), were treated with anti-Fas (CH-11 monoclonal antibody) to induce apoptosis.Cytotoxicity was detected by MTT assay. Symptoms of cell death were assessed by morphological observation after Hoechst33258 staining, activation of caspase-8 and caspase-3 was measured by Western blotting.RESULTS: Compared with KB cells, the resistance of KBv200 cells to VCR (vincristine) was about 51-fold higher.Anti-Fas (CH-11) induced cytotoxicity and apoptosis in both sensitive KB cells and MDR phenotype KBv200cells. The IC50 of CH-11 in KB cells was similar to that in KBv200 cells. CH-11 induced similar apoptotic rates in both KB cells and KBv200 cells, which could be classified as caspase-dependent apoptotic pathway. Verapamil (VRP) did not affect CH-11-mediated apoptosis in KBv200 cells.CONCLUSION: Expression of P-glycoprotein does not induce resistance to caspase-8 and -3 activation or antiFas-induced cell apoptosis.

Relationship between P-glycoprotein and CD44 expression in esophageal carcinoma

Objective： To investigate the relationship between P-glycoprotein （P-gp） and adhesion molecule CD44 expression as well as their clinical significance in esophageal carcinoma. Methods： To examine the expressed level of P-gp and CD44 by flow cytometry （FCM） in the operated samples of 70 cases with esophageal carcinoma and their normal mucosa of esophageal incision, and to evaluate their relationship with clinicopathological factors. Results： Among the 70 cases with esophageal carcinoma, the expression of P-gp in the 27 cases （38.6%） was negative （positive cells 〈25%）; 11 cases （15.7%） were 25%-40% expression of P-gp positive cells; 14 cases （20%） were 41%-60% expression of P-gp positive cells; 18 cases （25.7%） were the high expression （positive cells 〉60%） of P-gp. Of the cases with the tumor sizes being more than 4 cm, the expression of CD44 showed a significant difference （P〈0.05） in 25 cases with P-gp positive, compared with 19 cases with P-gp negative. Of the cases with high-mild differentiated esophageal carcinoma, the expression of CD44 showed a significant difference （P〈0.05） in 22 cases with P-gp positive, compared with 17 cases with P-gp negative. Of the cases with clinical Ⅲ-Ⅳ stage, the expression of CD44 showed a significant difference （P〈0.05） in 26 cases with P-gp positive, compared with 10 cases with P-gp negative. Of the cases with lymph node metastasis, the CD44 expression showed a significant difference （P=0.050） in 27 cases with P-gp positive, compared with 11 cases with P-gp negative. Of the cases of the patients＇ age being more than 56 years, the expression of CD44 showed a significant difference （P〈0.01） in 27 cases with P-gp positive, compared with 12 cases with P-gp negative. When the P-gp and CD44 expression were positive, the clinical Ⅱ stage and Ⅲ-Ⅳ stage in esophageal carcinoma was showed a significant difference （P〈0.05）. Conclusion： When the CD44 and P-gp both have the positive high expression, it will be significantly associated with the esophageal carcinoma progression and metastasis, so both were a positive expression in esophageal carcinoma, it might suggest a poor and unfavorable prognosis result.

Isolation of Prawn(Exopalaemon carinicauda) Lipopolysaccharide and β-1,3-Glucan Binding Protein Gene and Its Expression in Responding to Bacterial and Viral Infections

The pattern recognition proteins （PRPs） play a major role in immune response of crustacean to resist pathogens. In the present study, as one of PRPs, lipopolysaccharide and 13-1, 3-glucan binding protein （LGBP） gene in the ridge tail white prawn （Exopalaemon carinicauda） （EcLGBP） was isolated. The full-length cDNA of EcLGBP was 1338 bp, encoding a polypeptide of 366 amino acid residules. The deduced amino acid sequence of EcLGBP shared high similarities with LGBP and BGBP from other crus- taceans. Some conservative domains were predicted in EcLGBP sequence. EcLGBP constitutively expressed in most tissues at dif- ferent levels, and the highest expression was observed in hepatopancreas. With infection time, the cumulative mortality increased gradually followed by the proliferation of Vibrio parahaemolyticus and white spot syndrome virus （WSSV）. The expression of EcLGBP in response to E parahaemolyticus infection was up-regulated in hemocytes and hepatopancreas, and the up-regulation in hepatopancreas was earlier than that in hemocytes. EcLGBP expression after WSSV infection increased at 3 h, then significantly decreased in both hemocytes and hepatopancreas. The results indicated that EcLGBP was involved in the immune defense against bacterial and viral infections.

Surface Display of Rice Stripe Virus NSvc2 and Analysis of Its Membrane Fusion Activity

Rice stripe virus (RSV) infects rice and is transmitted in a propagative manner by the small brown planthopper. How RSV enters an insect cell to initiate the infection cycle is poorly understood. Sequence analysis revealed that the RSV NSvc2 protein was similar to the membrane glycoproteins of several members in the family Bunyaviridae and might induce cell membrane fusion. To conveniently study the membrane fusion activity of NSvc2, we constructed cell surface display vectors for expressing Nsvc2 on the insect cell surface as the membrane glycoproteins of the enveloped viruses. Our results showed that NSvc2 was successfully expressed and displayed on the surface of insect Sf9 cells. When induced by low pH, the membrane fusion was not observed in the cells that expressed NSvc2. Additionally, the membrane fusion was also not detected when co-expressing Nsvc2 and the viral capsid protein on insect cell surface. Thus, RSV NSvc2 is probably different from the phlebovirus counterparts, which could suggest different functions. RSV might enter insect cells other than by fusion with plasma or endosome membrane.

The expression of glucose regulated protein-94 in colorectal carcinoma cells treated by sodium butyrate

The expression of glucose regulated protein 94 (GRP94)during the treatment of human colorectal carcinoma cell lineClone A cells with sodium butyrate was studied. Sodium butyrate (SB) can cause functional and morphological effects on Clone A cells including growth arrest at Go/G1 stage and cell differentiation as observed by morphological changes, MTT and flow cytometry assays, as well as reduced Grp94 gene expression as shown by Northern blot and Western blot assays. The possible mechanism of the correlation between Grp94 gene expression and tumor growth inhibition and cell differentiation is briefly discussed.

Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum

Objective： Hepatocellular carcinoma （HCC） is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples. Methods： Lectin affinity chromatography （LAC） was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography （LC） separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins. Results： A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain （FGG）, FOS-like antigen 2 （FOSL2）, and a-l, 6-mannosylglycoprotein 6-^-N-acetylglucosaminyltransferase B （MGATSB） were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples. Conclusion： A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC.

Synthesis and anti-shock activity of divalent β-D-galactopyranosyl-(1→4)-β-D-glucopyranosides

Three divalent β-D-galactopyranosyl-（1 → 4）-β-D-glucopyranosides were synthesized using acetylated lactosyl bromide as donor and polyethylene glycols with different polymeric degree （n = 4, 5, 6） as linker, and evaluated for in vivo inhibitory activity to leukocyte-endothelial cell adhesion on severe bum-shock rats. The result showed that the length of linkers had apparent influence on anti cell adhesion activity.

Antidiabetic effects of chitooligosaccharides on pancreatic islet cells in streptozotocin-induced diabetic rats

AIM: To investigate the effect of chitooligosaccharides on proliferation of pancreatic islet cells, release of insulin and 2 h plasma glucose in streptozotocin-induced diabetic rats.METHODS: In vitro, the effect of chitooligosaccharides on proliferation of pancreatic islet cells and release of insulin was detected with optical microscopy, colorimetric assay, and radioimmunoassay respectively. In vivo, the general clinical symptoms, 2 h plasma glucose, urine glucose, oral glucose tolerance were examined after sixty days of feeding study to determine the effect of chitooligosaccharides in streptozotocin-induced diabetic rats. RESULTS: Chitooligosaccharides could effectively accelerate the proliferation of pancreatic islet cells. Chitooligosaccharides (100 mg/L) had direct and prominent effect on pancreastic β cells and insulin release from islet cells. All concentrations of chitooligosaccharides could improve the general clinical symptoms of diabetic rats, decrease the 2 h plasma glucose and urine glucose, and normalize the disorders of glucose tolerance.CONCLUSION: Chitooligosaccharides possess various biological activities and can be used in the treatment of diabetes mellitus.

Membrane glycoprotein M6A promotes μ-opioid receptor endocytosis and facilitates receptor sorting into the recycling pathway

The interaction of p-opioid receptor （MOPr） with the neuronal membrane glycoprotein M6a is known to facilitate MOPr endocytosis in human embryonic kidney 293 （HEK293） cells. To further study the role of M6a in the post-endocytotic sorting of MOPr, we investigated the agonist-induced co-internalization of MOPr and M6a and protein targeting after internalization in HEK293 cells that co-expressed HA-tagged MOPr and Myc-tagged M6a. ,We found that M6a, MOPr, and Rab 11, a marker for recycling endosomes, co-localized in endocytotic vesicles, indicating that MOPr and M6a are primarily targeted to recycling endosomes after endocytosis. Furthermore, co-expression of M6a augmented the post-endocytotic sorting of 6-opioid receptors into the recycling pathway, indicating that M6a might have a more general role in opioid receptor post-endocytotic sorting. The enhanced post-endocytotic sorting of MOPr into the recycling pathway was accompanied by a decrease in agonist-induced receptor down-regulation of M6a in co-expressing cells. We tested the physiological relevance of these findings in primary cultures of cortical neurons and found that co-expression of M6a markedly increased the translocation of MOPrsfrom the plasma membrane to intracellular vesicles at steady state and significantly enhanced both constitutive and agonist-induced receptor endocytosis. In conclusion, our results strongly indicate that M6a modulates MOPr endocytosis and post-endocytotic sorting and has an important role in receptor regulation.

CONSTRUCTING ADENO-ASSOCIATED VIRUS-TGFβ_3 AND COMPARING ITS BIOLOGICAL EFFECT ON PROTEOGLYCAN SYNTHESIS IN DEDIFFERENTIATED NUCLEUS PULPOUS CELLS WITH ADENOVIRUS-TGFβ_1

Objective To construct adeno-associated virus (AAV) expression system for transforming growth factor β3 (TGFβ3) and detect its biological effect on proteoglycan synthesis of the earlier and later dedifferentiated rabbit lumbar disc nucleus pulpous (NP) cells, which was compared with that of adenovirus (AV) expression system for TGFβ1. Methods TGFβ3 gene was obtained using PCR. Its upstream contained restriction enzyme site Kpn Ⅰ, and its downstream contained restriction enzyme site SalⅠ. Using the restriction enzyme sites of PCR product of TGFβ3 and the corresponding multiple cloning site (MCS) in plasmid AAV, TGFβ3 was subcloned into AAV. The recombinant plasmid AAV-TGFβ3 was transfected into H293 cells with LipofectamineTM 2000, and the expression of TGFβ3 gene was detected using immunofluorescent analysis. After AAV-TGFβ3 virus particle with infectious activity was packaged, TGFβ3 expression in NP cells was detected by immunoblotting, and its biological effect on proteoglycan synthesis was detected by antonopulos method and compared with that of AV-TGFβ1 in the earlier and later dedifferentiated NP cells. Results For the earlier dedifferentiated NP cells, AAV-TGFβ3 slowly and stably enhanced proteoglycan synthesis, but AV-TGFβ1 rapidly and transiently enhanced its synthesis. For the later dedifferentiated NP cells, AAV-TGFβ3 stably enhanced proteoglycan synthesis, but AV-TGFβ1 inhibited its synthesis. Conclusion AAV expression system can mediate TGFβ3 gene to be expressed stably, and AAV-TGFβ3 can enhance proteoglycan synthesis of the earlier and later dedifferentiated NP cells.