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Objective To explore the effect of laparoscopic salpingectomy on ovarian reserve function and serum neuropeptide Y(NPY)and cortisol(Cor)in patients with ectopic pregnancy.Methods 82 patients with ectopic pregnancy who had visited Chaohu Hospital Affiliated to Anhui Medical University from June 2018 to June 2021 were randomly divided into two groups,with 41 in each group.The control group underwent laparoscopic salpingostomy and suturing for embryo retrieval,while the study group underwent laparoscopic salpingectomy.Surgical duration,intraoperative blood loss,time to postsurgical mobilization,length of hospital stay,serum NPY and Cor levels,antral follicle count(AFC),indexes for peak flow velocity and resistance of ovarian artery were compared between the two groups.Follow-up was conducted for two years after surgery,and the success rate of pregnancy and the ectopic pregnancy rate were recorded.Results The surgical duration in the study group was shorter than that in the control group(P<0.05).The differences in serum NPY and Cor values between the two groups immediately after surgery and at postoperative week one were not statistically significant(P>0.05).At postoperative months1 and 6,the peak flow velocity of the affected ovary was lower in the study group than in the control group,whereas the resistance index was higher(P<0.05).At postoperative month 6,both groups showed an increase in peak flow velocity and a decrease in resistance index(P<0.05).There were no statistically signifi-cant differences in AFC and the success rate of pregnancy between the two groups postoperatively(P>0.05).The ectopic pregnancy rate in the study group was lower than that in the control group(P<0.05).Conclusion As compared with laparoscopic salpingostomy,laparoscopic salpingectomy for ectopic pregnancy can also preserve postoperative ovarian antral follicle count(AFC),and it has greater advantage in reducing the risk of recurrent ectopic pregnancy after surgery.
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There are some limitations of traditional influenza vaccines concerning novel mutant strains. Therefore, it is particularly important to develop preventive means for antigen-unrelated types of influenza viruses. Recent studies have shown that probiotics can modulate the immune system and reduce the severity of viral infections. In this study, we investigated the potential of Lactiplantibacillus plantarum 0111 against influenza virus H9N2. Challenge experiments showed that L. plantarum 0111 pretreatments could effectively improve mice's survival rate and weight loss and reduce the inflammatory cytokines IL-6 and TNF-α in the lungs and bronchoalveolar lavage fluid (BALF) along with the degree of lung and intestinal injury. FMT experiment demonstrates that the protective effect produced by L. plantarum 0111 is associated with gut microorganisms. In addition, 16S high-throughput sequencing of the mouse intestinal microbiota showed that L. plantarum 0111 remodeled the intestinal microbiota after H9N2 infection and maintained the gut microbiota balance. In a mouse model, the oral administration of L. plantarum 0111 increased IFN-ß expression in the serum and BALF. At the same time, the transcript levels of IFN-ß and related ISGs in the intestine and lungs of mice were also increased. In addition, the activation and polarization of T cells in mesenteric lymph nodes (MLNs) and the spleen were detected by flow cytometry, and the results showed that L. plantarum 0111 modulated cytokines in T cells and increased IgA expression in B cells in the MLNs and spleen. Thus, L. plantarum 0111 may improve gut microbiota-mediated immune responses and thus, resist infection by the influenza virus, and it could be used as an effective preventive measure against the influenza virus.
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BACKGROUND: Viral tracers are important tools for mapping brain connectomes. The feature of predominant anterograde transneuronal transmission offers herpes simplex virus-1 (HSV-1) strain H129 (HSV1-H129) as a promising candidate to be developed as anterograde viral tracers. In our earlier studies, we developed H129-derived anterograde polysynaptic tracers and TK deficient (H129-dTK) monosynaptic tracers. However, their broad application is limited by some intrinsic drawbacks of the H129-dTK tracers, such as low labeling intensity due to TK deficiency and potential retrograde labeling caused by axon terminal invasion. The glycoprotein K (gK) of HSV-1 plays important roles in virus entry, egress, and virus-induced cell fusion. Its deficiency severely disables virus egress and spread, while only slightly limits viral genome replication and expression of viral proteins. Therefore, we created a novel H129-derived anterograde monosynaptic tracer (H129-dgK) by targeting gK, which overcomes the limitations of H129-dTK. METHODS: Using our established platform and pipeline for developing viral tracers, we generated a novel tracer by deleting the gK gene from the H129-G4. The gK-deleted virus (H129-dgK-G4) was reconstituted and propagated in the Vero cell expressing wildtype H129 gK (gKwt) or the mutant gK (gKmut, A40V, C82S, M223I, L224V, V309M), respectively. Then the obtained viral tracers of gKmut pseudotyped and gKwt coated H129-dgK-G4 were tested in vitro and in vivo to characterize their tracing properties. RESULTS: H129-dgK-G4 expresses high levels of fluorescent proteins, eliminating the requirement of immunostaining for imaging detection. Compared to the TK deficient monosynaptic tracer H129-dTK-G4, H129-dgK-G4 labeled neurons with 1.76-fold stronger fluorescence intensity, and visualized 2.00-fold more postsynaptic neurons in the downstream brain regions. gKmut pseudotyping leads to a 77% decrease in retrograde labeling by reducing axon terminal invasion, and thus dramatically improves the anterograde-specific tracing of H129-dgK-G4. In addition, assisted by the AAV helper trans-complementarily expressing gKwt, H129-dgK-G4 allows for mapping monosynaptic connections and quantifying the circuit connectivity difference in the Alzheimer's disease and control mouse brains. CONCLUSIONS: gKmut pseudotyped H129-dgK-G4, a novel anterograde monosynaptic tracer, overcomes the limitations of H129-dTK tracers, and demonstrates desirable features of strong labeling intensity, high tracing efficiency, and improved anterograde specificity.
Subject(s)
Herpesvirus 1, Human , Animals , Axons , Brain , Herpesvirus 1, Human/genetics , Mice , NeuronsABSTRACT
Objective: To investigate the regulation of autophagy by SUMO specific peptidase 3 (SENP3, normally called SUMO specific protease 3) in mouse testis. Methods: Immunofluorescence was used to detect the localization of SENP3 in spermatogenic cells and Sertoli cells of testis. Senp3 wild type (Senp3+/+) mice and Senp3 gene knockout heterozygous (Senp3+/-) mice were subjected to starvation treatment to induce autophagy. Testicular tissue proteins were extracted, and the extent of autophagy was detected by Western blotting. The extent of autophagy of Sertoli cells was detected and compared with that of spermatogenic cells in testis by transmission electron microscopy and immunofluorescence. Results: SENP3 mainly localized in the nucleus of Sertoli cells. Compared to Senp3+/+ mice, the extent of starvation-induced autophagy in Sertoli cells of Senp3+/- mice increased. Conclusion: SENP3 can inhibit autophagy in Sertoli cells during nutrient deficiency, which may play a role in controlling the extent of autophagy.
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Clinical interpretation of the test results for cortisol based on continuous reference intervals with appropriate partitions improves pediatric diagnosis; however, these values are available only for Caucasians. To develop the pediatric reference intervals for Chinese population, we examined the serum cortisol levels in 1,143 healthy Chinese children aged 4–18 years (566 boys and 577 girls), using an IMMULITE 2000 Immunoassay System (Siemens Healthcare GmbH). Phlebotomy was performed at 7–9 a.m. for 284 boys and 287 girls and at 1–3 p.m. for the others. They were divided into four age groups according to the Clinical and Laboratory Standards Institute guideline EP28-A3c, with the last group further stratified according to sampling time. Separate reference intervals of 49.6–323.7, 70.9–395.3, and 90.1–448.7 nmol/L were established for children aged 4–8, 9–12, and 13–15 years, respectively. Further, reference intervals of 118.2–464.7 and 71.4–446.7 nmol/L were established for morning and afternoon cortisol levels, respectively, in children aged 16–18 years. Further studies are necessary to transfer and validate these reference intervals in other analytical systems and pediatric populations, and to allow for broader applications.
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Child , Female , Humans , Asian People , Delivery of Health Care , Diagnosis , Hydrocortisone , Immunoassay , Pediatrics , PhlebotomyABSTRACT
In 2016, a severe leaf spot disease was found on Iris ensata Thumb. in Nanjing, China. The symptom was elliptical, fusiform, or irregularly necrotic lesion surrounded by a yellow halo, from which a small-spored Alternaria species was isolated. The fungus was identified as Alternaria iridiaustralis based on morphological characteristics. The pathogenicity tests revealed that the fungus was the causal pathogen of the disease. Phylogenic analyses using sequences of ITS, gpd, endoPG, and RPB2 genes confirmed the morphological identification. This study is the first report of A. iridiaustralis causing leaf spots on I. ensata in China.
Subject(s)
Alternaria , China , Fungi , Iris , Sequence Analysis , Thumb , VirulenceABSTRACT
Transcription factor cAMP response element-binding protein (CREB) plays a critical role in memory formation. Ubiquitin-proteasome system-dependent protein degradation affects the upstream signaling pathways which regulate CREB activity. However, the molecular mechanisms of proteasome inhibition on reductive CREB activity are still unclear. The current study demonstrated that MG132-inhibited proteasome activity resulted in a dose dependence of CREB dephosphorylation at Ser133 as well as decreased phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunit NR2B (Tyr1472) and its tyrosine protein kinase Fyn (Tyr416). These dephosphorylations are probably caused by disturbance of expression and post-translational modifications of tau protein since tau siRNA decreased the activity of Fyn, NR2B, and CREB. To further confirm this perspective, HEK293 cells stably expressing human tau441 protein were treated with MG132 and dephosphorylations of CREB and NR2B were observed. The current research provides an alternative pathway, tau/Fyn/NR2B signaling, regulating CREB activity.
Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , Protein Processing, Post-Translational , Receptors, N-Methyl-D-Aspartate/metabolism , tau Proteins/metabolism , Animals , Cell Line, Tumor , HEK293 Cells , Humans , Leupeptins/pharmacology , Mice , Neurons/drug effects , Neurons/metabolism , Phosphorylation , Proteasome Inhibitors/pharmacology , Signal TransductionABSTRACT
Resistance to taxol represents a major obstacle for long-term remission in ovarian cancer. Transforming Growth Factor-ß-Activated Kinase 1 (TAK1) is a critical component in immune response pathway. However, the role of TAK1 in the development of chemoresistance in ovarian cancer remains unknown. Here, we showed that in vitro, taxol-resistant cells expressed higher TAK1, and the ratio of p-TAK1/TAK1 positively associated with taxol resistance in ovarian cancer cells. Inactivation of TAK1 by inhibitor 5Z-7-oxozeaenol or gene knockdown sensitized taxol cytotoxicity in vitro, promoting cell apoptosis and mitosis arrest. Moreover, resistant cells were much more sensitive to the combined TAK1 inhibitor and taxol treatment than their parental counterparts. Using xenograft mouse model, we found that 5Z-7-oxozeaenol significantly enhanced taxol efficacy in vivo. Thus, targeting TAK1 pathway is a promising strategy to enhance taxol response in ovarian cancer treatment.
Subject(s)
MAP Kinase Kinase Kinases/metabolism , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/enzymology , Paclitaxel/therapeutic use , Animals , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Enzyme Activation/drug effects , Female , Gene Knockdown Techniques , Humans , Mice , Neoplasm Recurrence, Local/enzymology , Neoplasm Recurrence, Local/pathology , Ovarian Neoplasms/pathology , Paclitaxel/pharmacology , Phosphorylation/drug effects , Treatment Outcome , Xenograft Model Antitumor Assays , Zearalenone/analogs & derivatives , Zearalenone/pharmacologyABSTRACT
The rare N-unsubstituted glucosamine (GlcNH3(+)) residues in heparan sulfate (HS) have important biological and pathophysiological roles. In this study, a high-resolution method for the separation and analysis of N-unsubstituted disaccharides of heparin/HS is described. Four N-unsubstituted disaccharides, together with eight N-substituted species, can be well-separated by ion-pair reverse-phase ultra-performance liquid chromatography. Each disaccharide can then be detected and its relative abundance quantified using electrospray ionization mass spectrometry in the negative mode. Because of its high sensitivity, without interference from proteins and other sample impurities, this method is particularly useful in the analysis of low content GlcNH3(+) residues in small amounts of biological materials, eg. sera, tissue and cell culture-derived samples. This would lead to a better understanding of the biological origin of GlcNH3(+) residues and their increasingly important function in human health and disease.
Subject(s)
Anticoagulants/chemistry , Disaccharides/analysis , Heparin/chemistry , Heparitin Sulfate/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Animals , Cattle , Chromatography, High Pressure Liquid/methods , Humans , SwineABSTRACT
The rare N-unsubstituted glucosamine (GlcNH3(+)) residues in heparan sulfate (HS) have important biological and pathophysiological roles. Therefore, the ability to chemically generate a series of oligosaccharides, which have a similar structure to the naturally-occurring, GlcNH3(+)--containing oligosaccharides from HS, would greatly contribute to investigating their natural role in HS. In this study, a hexasaccharide library that possess GlcNH3(+) residues were prepared from the chemical modification of the fully sulfated dp6. Chemical reaction conditions were optimized to generate different pattern of GlcNH3(+)--containing oligosaccharides, then the structure of the library was detected by high-performance liquid chromatography-ion trap/time-of-flight mass spectrometry (LC/MS-ITTOF) analysis. EIC/MS and MS(2) analysis showed different fragmentation patterns of dp6s with different GlcNH3(+) residues. This provides a foundation for further identification and quantification of GlcNH3(+)--oligosaccharides by mass spectrum analysis.
Subject(s)
Glucosamine/chemistry , Oligosaccharides/chemical synthesis , Chromatography, High Pressure Liquid , Glycosides/chemistry , Heparin/chemistry , Heparitin Sulfate/chemistry , Hexosamines/chemistry , Molecular Weight , Oligosaccharides/chemistry , Spectrometry, Mass, Electrospray Ionization , Tandem Mass SpectrometryABSTRACT
The rare N-unsubstituted glucosamine (GlcNH(3)(+)) residues in heparan sulfate (HS) have important biological and pathophysiological roles. Because of their low natural abundance, the use of chemically generated, structurally defined, N-unsubstituted heparin/HS oligosaccharides can greatly contribute to the investigation of their natural role in HS. However, the sequencing of mixtures of chemically generated oligosaccharides presents major challenges due to the difficulties in separating isomers and the available detection methods. In this study, we developed and validated a simple and sensitive method for the sequence analysis of N-unsubstituted heparin/HS oligosaccharides. This protocol involves pH 4 nitrous acid (HNO(2)) degradation, size-exclusion HPLC and ion-pair reversed-phase liquid chromatography-ion trap/time-of-flight mass spectrometry (IPRP-LC-ITTOF MS). We unexpectedly found that absorbance at 232 nm (normally used for specific detection of C4-C5 unsaturated oligosaccharides) was, in most cases, still sufficiently sensitive to also simultaneously detect saturated oligosaccharides during HPLC, thus simplifying the positional analysis of GlcNH(3)(+)) residues. The IPRP-LC-ITTOF MS system can supply further structural information leading to full sequence determination of the original oligosaccharide. This new methodology has been used to separate and sequence a variety of chemically generated, N-unsubstituted dp6 species containing between 1 and 3 GlcNH(3)(+)) residues per oligosaccharide in different positional combinations. This strategy offers possibilities for the sequencing of natural N-unsubstituted oligosaccharides from HS and should also be applicable, with minor modification, for sequencing at N-sulfated residues using alternative pH 1.5 HNO(2) scission.
Subject(s)
Heparin/chemistry , Heparitin Sulfate/chemistry , Oligosaccharides/chemistry , Carbohydrate Sequence , Chromatography, Gel , Chromatography, High Pressure Liquid , Mass Spectrometry , Molecular Sequence DataABSTRACT
Objective To explore the magnetic resonance angiography (MRA),digital subtraction angiography (DSA) imaging characteristics of blood stream related aneurysms associated with Moyamoya disease and its treatment method.Methods Retrospective analysis was carried out on the clinical data of 6 patients with aneurysms related to Moyamoya disease,admitted to our hospital from January 2000 to September 2012.Results Among the 6 patients,3 had ischemic cerebrovascular disease with un-ruptured aneurysm; 2 of the 3 patients with ruptured aneurysm manifested as subarachnoid hemorrhage and 1 as hemorrhage in the basal ganglia.Three patients had major artery aneurysms around Willis circles,2 was peripheral artery type,and 1 was the mixed type.Endovascular embolization was performed in 4 patients and the other 2 accepted conservative treatments.Follow-up results were satisfied.Conclusion Blood stream related aneurysms associated with Moyamoya disease can be detected by MRA and DSA; endovascular embolization for major artery aneurysms is safe and effective and patients with peripheral artery type aneurysm not having hemorrhage,having surgical difficulty,can be treated with conservative treatment and performed follow up regularly.
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<p><b>OBJECTIVE</b>To investigate the relationship between HBV DNA and hepatitis B virus large envelope protein (HBV-LP) in patients with chronic hepatitis B.</p><p><b>METHODS</b>Serum HBV DNA was detected by RT-PCR and the HBV-LP was detected by enzyme linked immunosorbent assay in 320 serum samples collected from patients with chronic hepatitis B.</p><p><b>RESULTS</b>There were no significant difference between positive rate of HBV-LP and that of HBV DNA in different HBeAg patterns (P>0.05). Serum HBV-LP levels were closely correlated with HBV DNA copies (r=0.949).</p><p><b>CONCLUSION</b>Serum HBV-LP is a reliable serological marker that can reflect the replication of HBV.</p>
Subject(s)
Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , DNA, Viral , Blood , Hepatitis B virus , Physiology , Hepatitis B, Chronic , Blood , Virology , Viral Envelope Proteins , Blood , Virus ReplicationABSTRACT
Objective To investigate the mechanism responsible for the malignant progressionof meningiomas at the protein level using tissue microarray technique. Methods Twenty-twointracranial meningioma tissue microarrays were constructed, each containing the tissues of 42 benign, 18atypical, and 19 anaplastic meningiomas. Immunohistochcmistry of the microarrays was performed induplicate with the antibodies of MYC, ARNT2, MDM2, AR, ER, PR, Ki-67, P53, survivin, CD34 andVEGF, respectively. Negative control microarrays were used throughout the experiment and breast cancertissue microarrays were used as the positive controls for ER and PR staining. SAS9.0 solfware was usedfor grading of the expression levels of the biomarkers according to the WHO grades of meningiomas.Results For each antibody, the duplicate tissue microarrays yielded uniform staining results invisualization of the protein distributions in the cytoplasm and nuclei, and the negative controls displayedno positive staining. The p53, AR, ER, PR and Ki-67 proteins were found only in the cell nuclei, MDM2in both the cytoplasm and nuclei, and ARNT2, CD34 and VEGF in the cytoplasm only. The c-MYC andsurvivin proteins were found mainly in the cytoplasm, and in some instances in both the cytoplasm andcell nuclei. Immunohistochemical staining for p53, AR, CD34, Ki-67 and MYC proteins showed strongcorrelations to the degree of malignancy of the meningioma (P<0.05). Conclusions Tissue microarrayand immunohistochemical techniques provide an efficient means for screening the specific biomatkers ofmeningiomas. The expressions of p53, AR, CD34, Ki-67 and MYC proteins are involved in the malignantprogression of meningioma, and these proteins may serve as important biomarkers for meningiomagrading at the protein level.
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0.05)in the frequency of alleles and genotypes between controls and coronary heart disease.In additional,at the 325 position,the TAFI antigen of the Thr325Thr was higher[(114.89?2.53)%]than that of the other genotype(Thr325Ile and Ile325Ile),there was significant difference between the TAFI antigen of the Thr325Thr and the others(P 0.05).But the TAFI activity of the Ile325Ile was lower(3.08?3.63 ?g/ml)than that of the other genotypes(Thr325Ile and Thr325Thr),there was significantly difference between the TAFI activity of the Thr325Thr and the other(P
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A main complication of chemotherapy in cancer patients is hematopoiesis suppression. Microenviroment transplantation using bone marrow stromal cells (BMSCs) has been demonstrated to be a potent method in recovery of hematopoiesis in animal models. Based on hematopoiesis-supportive ability of BMSCs and high potency of IL-3 in hematopoiesis stimulation, BMSCs were studied as a cellular delivery system for IL-3 gene transfection to promote hematopoiesis recovery of mice after high dose chemotherapy. BMSCs were transfected with recombinant adenovirous containing murine IL-3 gene(MOI = 10), the level of mIL-3 secreted by gene-modified BMSCs was 110U/ml/10~6 cells/ 24h in vitro. The mice were injected with high dose cyclophosphamide(200mg/kg) i.p. and after 24 hours the IL-3 gene-modified BMSCs(2 x 10~6/mouse) were transplanted intrasplenically. White blood cell counts in peripheral blood of mice received intrasplenic injection of IL-3-BMSCs were kept at a high level within two weeks after chemotherapy. The pathological sections of spleens and bone marrow showed significant recovery of hematopoiesis, compared with that of mice received chemotherapy only. The data indicated the feasibility of IL-3 gene-modified BMSCs transplantation in the acceleration of hematopoiesis recovery after chemotherapy.
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Adenoviruses harboring E. coli cytosine deaminase gene (AdCD) were used to transfect murine FBL-3 ery-throleukemia cells in vitro. FBL3 cells infected with AdCD were more sensitive to 5-fluorocytosine (5-FC) than cells infected with a control adenovirus AdLacZ. Further study indicated that this combination therapy (AdCD and 5-FC) killed tumor cells by inducing apoptosis of FBL-3 cells. The supematants from FBL-3 cells treated with AdCD/5-Fc were transferred on the culture system of uninfected (wild - type) FBL-3 cells, the result indicated that only 6.25% of the supernatant could induce significant cytotoxicity on wild type FBL3 cells. The results demonoustrated that bystander effect plays an important role in AdCD-mediated cytotoxicities. Direct injection of AdCD into established subcutaneous FBL3 tumor in mice followed by daily intraperitoneal injection of 5-FC for 10 days was found to inhibit tumor growth significant-
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Antitumor effect of combined transfer of suicide gene and cytokine gene was evaluated in the present study. Adenoviruses expressing E. coli. cytosine deaminase (AdCD) and adenoviruses expressing murine interleukin 2 (AdTL2) were used for the treatment of tumor-bearing mice. The mice were inoculated s. c. with FBL-3 leukemia cells and 3 days later received intratumoral injection of AdCD in the presence or absence of AdIL2 followed by intraperitoneal 5-fluorocytosine (5FC) administration. The results demonstrated that tumor-bearing mice treated with AdCD/5FC in combination with AdTL2 showed more .potent inhibition of tumor growth and survived much longer as compared with mice treated with AdCD/5FC, AdEL2, AdlacZ/5FC or PBS. It was illustrated that the tumor mass showed obvious necrosis and inflammatory cell infiltration, and more CD4+ and CD8+ T cells infiltrated into the tumor after combined therapy. The splenic NK and CTL activities increased significantly in mice after combined transfer of CD gene and EH gene. Our results demonstrated that combined transfer of suicide gene and IL-2 gene could inhibit the growth of established tumor in mice significantly and induce antitumor immunity of the host efficiently.
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Adenovirus harboring E. coli. cytosine deaminase gene (AdCD) and adenovirus encoding with lymphotactin gene (AdLtn) were used for gene therapy in vivo. BALB/c mice were inoculated subcutaneously with CT26 colon adeno-carcinoma cells and 3 days later received combined injection of AdCD and/or AdLtn followed by continuous injection with 5-fluorocytosine(5-FC) 300mg/kg. The results demonstrated that mice received combined therapy developed tumors most slowly and survived longest when compared with mice treated with AdCD/5-FC, AdLtn, AdlacZ/5-FC or PBS. To further explain the immunological mechanism of the antitumor effects by the combined therapy, we found that combined treatment with suicide gene and Ltn gene therapy achieved maximal cytotoxic effects of nature killer cells and specific cy-totoxic T lymphocytes. FACS analysis of the tumor mass demonstrated that AdCD/5-FC in combination with AdLtn therapy increased the expression of H-2K~d and B7-1 expression on tumor cells. The CD4~+ and CD8~+ cells infiltrated in the tumor mass after combined therapy were significantly increased when measured by FACS analysis. Our results demonstrated that combined transfer of suicide gene and lymphotactin gene induce nonspecific and specific antitumor immunity of the host and elicit more potent antitumor effect.
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The hG -CSF cDNA was cloned by RT-PCR and confirmed by sequencing, which contains the full length of hG-CSF encoding region and parts of 5 '、3' non - coding region. Then the hG - CSF retroviral vector pLGSN was constructed by orientationally inserting the hG-CSF cDNA into the EcoRI/XhoI cloning sites of pLXSN vector. Packaged with CRE and CRIP packaging cell lines which are considered to be unlikely to produce helper viruses, the final pLGSN retrovirion titer reached 1. 1 ?106 CFU/ml. During constitutive passaging, the CRIP - LGSN cell clone produced relatively stable tilers of pLGSN retrovirion, ranging from 6. 8?105CFU/ml to 1. 1?106CFU/ml. By infecting the murine fibroblast cell line NIH3T3 with pLGSN retrovirion, a cell clone designated as NIH3T3 -G -CSF was obstained, secreting 168U/ml G-CSF . The integration and expression of hG-CSF gene in this cell clone were confirmed by Southern and Northern blotting analyses. Western blotting has also detected specifically the hG-CSF protein in the condensed supernalants from NIH3T3-G-CSF cells . After packaging the hG-CSF-secreting fibroblasts with collagen and implanting them into synergenic mice peritoneally , we detected a certain levels of G-CSF in the sera of mice, which suggested the implanted NIH3T3-G-CSF fibroblast cells could constitutively express and release hG-CSF in vivo. Our data showed the constructed hG-CSF retroviral vector could be used to further investigate the fibroblasl-mediated hG-CSF gene therapy .