ILC2 influence the differentiation of alveolar type II epithelial cells in bronchopulmonary dysplasia mice.

Bronchopulmonary dysplasia, a common complication of premature infants, is mainly characterized by blocked alveolarization. Proverbially, the injury of alveolar type II epithelial cells is regarded as the pathologic basis of occurrence and development of bronchopulmonary dysplasia. In the case of alveolar epithelial damage, alveolar type II epithelial cells can also differentiate to alveolar type I epithelial cells as progenitor cells. During bronchopulmonary dysplasia, the differentiation of alveolar type II epithelial cells becomes abnormal. Group 2 innate lymphoid cells can produce type 2 cytokines in response to a variety of stimuli, including the epithelial cytokines IL-25, IL-33, and thymic stromal lymphopoietin. Previous studies have shown that group 2 innate lymphoid cells can inhibit the alveolarization process of bronchopulmonary dysplasia by secreting IL-13. However, whether group 2 innate lymphoid cells can affect the differentiation of alveolar type II epithelial cells in the pathologic process of bronchopulmonary dysplasia remains unclear. In this study, we have shown that IL-13 secreted by group 2 innate lymphoid cells increased during bronchopulmonary dysplasia, which was related to the release of large amounts of IL-33 by impaired alveolar type II epithelial cells. This led to abnormal differentiation of alveolar type II epithelial cells, reduced differentiation to alveolar type I epithelial cells, and increased transdifferentiation to mesenchymal cells through the epithelial-mesenchymal transition. Taken together, our study provides a complementary understanding of the development of bronchopulmonary dysplasia and highlights a novel immune mechanism in the pathogenesis of bronchopulmonary dysplasia.

Sustained over-expression of calpain-2 induces age-dependent dilated cardiomyopathy in mice through aberrant autophagy.

Calpains have been implicated in heart diseases. While calpain-1 has been detrimental to the heart, the role of calpain-2 in cardiac pathology remains controversial. In this study we investigated whether sustained over-expression of calpain-2 had any adverse effects on the heart and the underlying mechanisms. Double transgenic mice (Tg-Capn2/tTA) were generated, which express human CAPN2 restricted to cardiomyocytes. The mice were subjected to echocardiography at age 3, 6, 8 and 12 months, and their heart tissues and sera were collected for analyses. We showed that transgenic mice over-expressing calpain-2 restricted to cardiomyocytes had normal heart function with no evidence of cardiac pathological remodeling at age 3 months. However, they exhibited features of dilated cardiomyopathy including increased heart size, enlarged heart chambers and heart dysfunction from age 8 months; histological analysis revealed loss of cardiomyocytes replaced by myocardial fibrosis and cardiomyocyte hypertrophy in transgenic mice from age 8 months. These cardiac alterations closely correlated with aberrant autophagy evidenced by significantly increased LC3BII and p62 protein levels and accumulation of autophagosomes in the hearts of transgenic mice. Notably, injection of 3-methyladenine, a well-established inhibitor of autophagy (30 mg/kg, i.p. once every 3 days starting from age 6 months for 2 months) prevented aberrant autophagy, attenuated myocardial injury and improved heart function in the transgenic mice. In cultured cardiomyocytes, over-expression of calpain-2 blocked autophagic flux by impairing lysosomal function. Furthermore, over-expression of calpain-2 resulted in lower levels of junctophilin-2 protein in the heart of transgenic mice and in cultured cardiomyocytes, which was attenuated by 3-methyladenine. In addition, blockade of autophagic flux by bafilomycin A (100 nM) induced a reduction of junctophilin-2 protein in cardiomyocytes. In summary, transgenic over-expression of calpain-2 induces age-dependent dilated cardiomyopathy in mice, which may be mediated through aberrant autophagy and a reduction of junctophilin-2. Thus, a sustained increase in calpain-2 may be detrimental to the heart.

C16, a novel sinomenine derivatives, promoted macrophage reprogramming toward M2-like phenotype and protected mice from endotoxemia.

Macrophage plays a critical part in host defense, tissue repair, and anti-inflammation; Macrophage reprogramming is responsible for disease development or regression. We aimed to clarify the effect of sinomenine-4-hydroxy-palmitate (C16), on macrophage reprogramming and anti-inflammatory in endotoxemia model. According to a structure modification of SIN (Sinomenine), C16 was found. Then, based on the endotoxin model, the mice liver and kidney toxicity was evaluated and serum cytokines level of IL-6 (Interleukin-6), TNF-α (Tumor necrosis factor-α), and IL-1ß (Interleukin-1ß) were measured by ELISA (Enzyme linked immunosorbent assay). Then, we confirmed the effect of C16 on macrophages reprogramming, we used the flow cytometry to test the effect of C16 on macrophages apoptosis in vitro. Then, iNOS (Inducible nitric oxide synthase), M1-type related cytokines, such as IL-1ß, TNF-α, and M2-type related cytokines, such as Arg-1 (Arginase-1), CD206, Fizz1, and Ym1 was detected, which expressed in ANA-1 and primary peritoneal macrophages. To further explore the molecular mechanism of C16 in reprogramming of macrophages from M1 toward M2 phenotype, the expression of STAT1 (signal transducer and activator of Transcription 1), STAT3, ERK1/2 (extracellular signal regulated kinase1/2), AKT, p38, and its corresponding phosphorylation were determined by western blot. Our results demonstrated that C16 improved the survival rate of LPS- (lipopolysaccharide) challenged mice and decreased the inflammatory cytokines expression; After C16 treatment, the expression of M1 phenotype correlation factors decreased significantly, while the expression of M2 phenotype correlation factors increased significantly at different levels compared with normal group. It indicated that C16 reprogram macrophages phenotype from M1 toward M2 following LPS stimulus. Furthermore, the results also showed that C16 showed anti-inflammatory effect by inhibiting LPS-induced p38, AKT and STAT1 phosphorylation and contributing ERK1/2 activation. C16 promoted macrophage reprogramming toward M2-like phenotype via p-p38/p-AKT or STAT1 signals pathway and C16 might be a valid candidate for inflammatory disease.

Gastric cancer tissue-derived mesenchymal stem cells impact peripheral blood mononuclear cells via disruption of Treg/Th17 balance to promote gastric cancer progression.

Gastric cancer tissue-derived mesenchymal stem cells (GC-MSCs) are important resident stromal cells in the tumor microenvironment (TME) and have been shown to play a key role in gastric cancer progression. Whether GC-MSCs exert a tumor-promoting function by affecting anti-tumor immunity is still unclear. In this study, we used GC-MSC conditioned medium (GC-MSC-CM) to pretreat peripheral blood mononuclear cells (PBMCs) from healthy donors. We found that GC-MSC-CM pretreatment markedly reversed the inhibitory effect of PBMCs on gastric cancer growth in vivo, but did not affect functions of PBMCs on gastric cancer cell proliferation, cell cycle and apoptosis in vitro. PBMCs pretreated with GC-MSC-CM significantly promoted gastric cancer migration and epithelial-mesenchymal transition in vitro and liver metastases in vivo. Flow cytometry analysis showed that GC-MSC-CM pretreatment increased the proportion of Treg cells and reduced that of Th17 cells in PBMCs. CFSE labeling and naïve CD4+ T cells differentiation analysis revealed that GC-MSC-CM disrupted the Treg/Th17 balance in PBMCs by suppressing Th17 cell proliferation and inducing differentiation of Treg cells. Overall, our collective results indicate that GC-MSCs impair the anti-tumor immune response of PBMCs through disruption of Treg/Th17 balance, thus providing new evidence that gastric cancer tissue-derived MSCs contribute to the immunosuppressive TME.

Expression of BMP4 in myocardium and vascular tissue of obese mice.

BACKGROUND: Obesity is regarded as a risk factor for cardiovascular disease. Bone morphogenetic protein 4 (BMP4) is a proinflammatory and profibrotic factor, and the reduced expression of this molecule in obese mice seems to be inconsistent with the known proinflammatory effects of obesity. Therefore, we studied BMP4 expression and inflammation in the myocardial tissue and aortas of obese mice. METHODS AND RESULTS: Four-week-old ob/ob mice were used as the experimental group, and C57BL/6 mice comprised the control group. Animals were sacrificed after a 12-week full diet, and then the blood, heart, abdominal aorta, and inguinal adipose tissue were collected. The expression of BMP4 mRNA and protein in the heart and aorta was significantly higher in the experimental group than in the control group, but expression was lower in adipose tissue. Inflammation measured by the expression of IL-1ß and IL-9 mRNA and protein and Smad1 and phosphorylated Smad1/5/8 protein in the heart and aorta was higher in the experimental group than in the control group. In addition, the expression of BMP4 in the serum was significantly higher in the experimental group than in the control group. CONCLUSION: BMP4 is significantly overexpressed in the myocardial tissue and aortas of obese mice, and mediates local inflammatory responses.

[Effect of IL-17 on collagen I/III expression in cardiac fibroblasts isolated from BALB/c mice].

AIM: To investigate the effect of IL-17 on the expression of collagen I/III in cardiac fibroblasts and analyze its molecular mechanism. METHODS: Cardiac fibroblasts were isolated from 7-14-day-old BALB/c mice and cultured in DMEM with 10% fetal bovine serum (FBS). The cells were collected after IL-17 treatment for 0, 24, 48, 72 h. IL-17 receptors on cardiac fibroblasts were detected by PCR; the collagen I/III expression was analyzed by immunofluorescence; the PKCß, Erk1/2, NF-κB phosphorylation were investigated by Western blotting. RESULTS: IL-17RA/C was expressed on cardiac fibroblasts; after 24 h of IL-17 stimulation, the collagen I/III expression obviously increased; Western blotting showed that PKCß, Erk1/2 and NF-κB were phosphorylated on 30, 45, 45 min, respectively. CONCLUSION: IL-17 could induce the expression of collagen I/III in cardiac fibroblasts, which might be related with PKCß-ERK1/2-NF-κB phosphorylation.

[Release of HMGB1 by LPS-treated cardiac fibroblasts and its contribution to the production of collagen type I and III].

AIM: To investigate whether cardiac fibroblasts (CFs) treated by LPS can actively secrete high-mobility group box protein 1 (HMGB1) and to analyze the correlation between HMGB1 releasing and the accumulation of collagen type I , III . METHODS: CFs were isolated from the heart of 7-14-day-old BALB/c mice and cultured in DMEM with 10% fetal bovine serum (FBS). We collected the CFs and cell supernatants after treated by LPS for 0, 6, 12, 24, 36, 48 h, respectively. The mRNA and protein expression levels of HMGB1, collagen 1a1 (col1a1) and collagen 3a1 (col3a1) in CFs after LPS stimulation were detected by RT-PCR and Western blotting, respectively. The intracellular localization of HMGB1 in treated CFs was investigated by immunofluorescence. RESULTS: After 0-6 h of LPS stimulation, the mRNA levels of HMGB1, col1a1, col3a1 had no significant changes; but increased obviously at 12, 24, 36, 48 h. HMGB1 was found in the cell supernatant by Western blotting after 24 h LPS stimulation, and its expression decreased following the first rise in CFs. Meanwhile, immunofluorescence showed HMGB1 translocation from nucleus to cytoplasm. The levels of col1a1 and col3a1 were up-regulated in CFs after stimulation. CONCLUSION: LPS can induce HMGB1 translocation from nucleus to cytoplasm and across cellular membrane to the outside of CFs at a time-dependent manner. Col1a1 and Col3a1, which are closely associated with myocardial fibrosis, were obviously up-regulated by LPS stimulation, which indicates that actively released HMGB1 might contribute to myocardial fibrosis following the endotoxin induced-sepsis.

[Construction, expression and immunogenicity of EV71 multiepitope-mGITRL eukaryotic plasmid].

AIM: To construct an enterovirus 71(EV71) multiepitope-mGITRL eukaryotic plasmid and study its immunogenicity in BALB/c mice. METHODS: We first designed and synthesized VP1' epigene containing two B cells and two T cells epitopes of VP1, and amplified mGITRL gene by PCR. The VP1' epigene and mGITRL gene were then cloned into the expression vector pIRES to construct the recombination plasmid pIRES-VP1'-mGITRL. The recombination plasmid was transfected into COS7 cells by liposome-mediated method. The protein expressions of VP1' and mGITRL were detected by Western blotting. BALB/c mice were immunized with pIRES-VP1'-mGITRL plasmid, and its serum antibody titer was measured by ELISA. RESULTS: The recombination plasmid pIRES-VP1'-mGITRL was successfully constructed as demonstrated by sequencing. Western blot analysis indicated that the VP1'-mGITRL fusion protein was expressed in COS7 cells and muscle cells. After BALB/c mice were immunized with this plasmid, we detected the high titer of anti-VP1 antibody in serum. CONCLUSION: VP1'-mGITRL fusion protein can be highly expressed in COS7 cells and muscle cells by the construction and transfection of the recombination plasmid pIRES-VP1'-mGITRL, and it could elicit the dramatic immune response in mice.

[Construction of eukaryotic co-expression vector of Porphyromonas gingivalis outer membrane protein ragB and glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL) and its immunogenic analysis].

AIM: To construct eukaryotic co-expression vector of Porphyromonas gingivalis outer membrane protein ragB and mouse glucocorticoid-induced tumor necrosis factor receptor ligand (mGITRL) and to analyze its immunogenicity in vivo. METHODS: The ragB gene was obtained from pMD18-T-ragB, and then cloned into the eukaryotic expression vector pIRES and pIRES-mGITRL, respectively. The eukaryotic expression vectors: pIRES-ragB and pIRES-ragB-mGITRL were identified by double enzyme digestion and DNA sequencing, then transfected into COS7 cells by Lipofectamine(TM);2000. The expressions of ragB or mGITRL in COS7 cells were detected by Western blotting. The mice were immunized with the recombinant pIRES-ragB-mGITRL plasmid. The serum antibody level was determined by ELISA. RESULTS: pIRES-ragB and pIRES-ragB-mGITRL plasmids were successfully constructed. Western blotting showed that the targeted gene was over-expressed in COS7 cells and skeletal muscle cells, respectively. The high titers of antibodies against RagB were detected in mouse serum. CONCLUSION: The construction of pIRES-ragB-mGITRL co-expression vector provides the experimental basis for Porphyromonas gingivalis vaccine research, prevention and treatment of periodontitis.

[The pathogenesis of CD4(+)T cells infiltrated into the spinal cord in rat SNL model].

AIM: To explore the infiltration pathogenesis of CD4(+);T cells following the spinal nerve ligation. METHODS: Healthy adult male SD rats were randomly divided into the spinal nerve ligation group (Tx), sham operation group (S), control group (C). the 50& mechanical paw withdrawal threshold ( 50&MWT ) was determined by up-down method; CD4(+);T cells infiltration was assessed by FACS; the mRNA levels of CCL2, CCL5 and CXCL10 were quantitated by RT-qPCR; serum cytokines were tested by ELISA kits. RESULTS: After 3 days since operation, 50&MWT of Tx group was significantly reduced (P<0.01) comparing with S group, C group; on day 14, 50&MWT was up to the minimum value; whereas S group and C group were no difference (P>0.05). After 7 days since operation, CD4(+);T cells infiltration into lumbar segments of the spinal cord in the Tx group increased significantly (P<0.01), and the CCL2, CCL5mRNA expression increased (P<0.05); on day 14, the CD4(+);T cells infiltration in Tx group was higher than S group, C group; but there was no statistical significance. On day 7 and 14 days, serum levels of cytokines were no difference in the three groups. CONCLUSION: Following spinal nerve ligation, high expression of chemokine promoted peripheral CD4(+);T cells to infiltrate into spinal cord; and the infiltrated CD4(+);T cells maintained the neuropathic pain.

Toll-like receptors are potential therapeutic targets in rheumatoid arthritis.

Toll-like receptors (TLRs) are found on the membranes of pattern recognition receptors and not only play important roles in activating immune responses but are also involved in the pathogenesis of inflammatory disease, injury and cancer. Furthermore, TLRs are also able to recognize endogenous alarmins released by damaged tissue and necrosis and/or apoptotic cells and are present in numerous autoimmune diseases. Therefore, the release of endogenous TLR ligands plays an important role in initiating and driving inflammatory diseases. Increasing data suggest a role for TLR signaling in rheumatoid arthritis, which is an autoimmune disease. Although their involvement is not comprehensively understood, the TLRs signaling transducers may provide potential therapeutic targets.

[Functional study of transcription factor Hlx modified dendritic cell line DC2.4].

AIM: To transfect Hlx into mouse dendritic cell line DC2.4 and observe the effect of hlx on function of dendritic cells. METHODS: The eukaryotic expression vector PIRES2-EGFP/Hlx was transfected into DC2.4 by liposomes. The transfection efficiency was identified through FACS. RT-PCR and Real-time PCR were used to test the transcription level of Hlx in DC2.4. Forty-eight hours after transfection, DC2.4 cells were studied for cytokine production, cell phenotype, phagocytosis, unilateral mixed lymphocyte reaction. RESULTS: The pIRES2-EGFP/Hlx vector was transfected into DC2.4 with the transfection efficiency of up to 60%. Highly expressed Hlx in DC2.4 increased the expression of maturation makers including CD80 and CD86, and major histocompatibility complex-II. Functional assay showed that over-expression of Hlx in DC2.4 increased the interleukin-12 transcription and decreased DC endocytosis. The Hlx modified DC2.4 highly expressed IL-10 and TGF-ß at the same time. Furthermore, it was shown that in a unilateral mixed lymphocyte reaction model, Hlx modified DC2.4 inhibited proliferation of lymphocytes. CONCLUSION: Transient over-expression of Hlx in DC2.4 promotes DC2.4 maturation and up-regulates IL-12, IL-10 and TGF-ß expression. However, the Hlx modified DC2.4 cells functionally appear as regulatory dendritic cells.

[The expression, identification of human HMGB1 B box and preparation of monoclonal antibodies against HMGB1 B box].

AIM: To express human HMGB1 B box protein and obtain monoclonal antibodies (mAbs) against HMGB1 B box for further study of the function of human HMGB1 protein. METHODS: pET28-HMGB1 B box plasmid transfected the DH5α, then expressed. And the extracted protein was purified by protein purification system. BALB/c mice were immunized with recombinant human HMGB1 B box protein. Hybridoma cell lines secreting mAb against human HMGB1 B box protein were screened by ELISA and subcloning approach. The characteristics of these mAbs were identified by ELISA and Western blot. RESULTS: Two hybridoma cell lines (1D2F4E3 and 2D4E3A2) stable secreting specific mAbs were successfully obtained.Western blot exhitited the two mAbs binded specifically to human HMGB1 B box protein. The immunoglobulin (Ig) class of two mAbs belonged to IgG, their titers were 1×10(6);, and the A(450); of mAb1D2F4E3, 2D4E3A2 were 0.324±0.093, 0.296±0.085, respectively. CONCLUSION: Two of high specificity mAbs against human HMGB1 B box protein have been successfully prepared, which laid the foundation for further study of biological function of human HMGB1 protein.

[Expression of TLR8 in human cervical cancer HeLa cells and the effect of TLR8 agonist on the cell proliferation and apoptosis].

OBJECTIVE: To observe the expression of Toll-like receptor 8 (TLR8) in human cervical cancer cell-line HeLa cells, and the effects of TLR8 agonist CL075 on the survival and proliferation of HeLa cells. METHODS: PCR and RT-PCR were used to detect the expression of TLR8 in 13 cancer cell lines, and the expression of COX-2, Bcl-2, VEGF mRNA in the HeLa cells stimulated by TLR8 agonist CL075 were also measured by RT-PCR. Immunofluorescence technique was used to determine the exact location of TLR8 in the cells. The percentage of viable cells was determined by trypan blue exclusion after the HeLa cells were stimulated with TLR8 agonist CL075 (0.1 µg/ml, 0.5 µg/ml, 1.0 µg/ml, 2.5 µg/ml), and cell cycle and apoptosis were analyzed by flow cytometry, and the proliferation was measured by MTT. RESULTS: Compared with the other cancer cell lines, the expression of TLR8 in HeLa cells was the highest (703.7 ± 20.6). After stimulation by CL075, the cells had a remarkable increase of the percentage of cells in G(2)/M + S phases. In the control group, the percentage of cells in G(2)/M +S phases was (39.02 ± 2.33)%, whereas after stimulated with 1.0 µg/ml CL075, the percentage of cells in G(2)/M + S phases reached the highest ratio (57.67 ± 1.73)%, and the percentage of cells in G(2)/M + S phases had a less decrease after 2.5 µg/ml CL075 stimulation and the percentage was (56.14 ± 3.73)%. After the CL075 treatment, there was no significant changes of apoptosis compared with that of the control cells (P > 0.05), but after DDP treatment the apoptosis had a significant change (P < 0.01). After stimulation by 1.0 µg/ml CL075 for 24 h, no significant difference (P > 0.05) was found by MTT test, but a significant difference was found at 48 h and 72 h (P < 0.01). An increased expression of COX-2, Bcl-2 and VEGF mRNA was observed in HeLa cells after stimulation by TLR8 agonist CL075 for 24 h and 48 h (P < 0.05). CONCLUSIONS: Expression of TLR8 is significantly increased in HeLa cells. The proportion of cells at different phases has a significant change after CL075 stimulation, which may up-regulate the proliferation of HeLa cells. These data suggested that TLR8 agonist may influence the tumor development and TLR8 may become a potential target in the treatment for cervical cancer.

[Detection and significance of transcription factors and cytokines of Th17/Treg cells in peripheral blood in the gastric cancer patients].

OBJECTIVE: To detect the expression levels of transcription factors and associated cytokines of Th17 and Treg cells in peripheral blood mononuclear cells (PBMC) of patients with gastric cancer, and explore the possible pathological mechanism of these cells involved in the development of gastric cancer. METHODS: The mRNA levels of RORgammat, FoxP3 in PBMC were determined by quantitative real-time PCR (QRT-PCR) from 57 patients with gastric cancer, 31 patients with benign gastric illness and 40 healthy people. The concentration of IL-17, IL-23, TGF-beta, IL-10 in plasma were detected by enzyme linked immunosorbent assay (ELISA). RESULTS: Compared with healthy volunteers, patients with gastric cancer showed higher levels of RORgammat and FoxP3 in PBMC (P < 0.05). The ratio of FoxP3/RORgammat in gastric cancer group was higher than that in the volunteer group and benign gastric illness group (P < 0.05). The ratio of FoxP3/RORgammat was higher in advanced disease than early disease (P < 0.05). The expressions of IL-17, IL-23, TGF-beta and IL-10 were higher in patients with gastric cancer than that in healthy volunteers (P < 0.05). In addition, The expression of TGF-beta and IL-10 were significantly increased in the advanced disease group than that in the early group (P < 0.05), but IL-17 and IL-23 was not significantly changed between the two groups (P > 0.05). CONCLUSION: There are higher levels of Th17 and Treg cells in gastric cancer patients, and it also shows a persistent predominant tendency of Treg cells and a reduced tendency of Th17 cells in advanced disease. Detecting the expression of Th17/Treg transcription factor and related cytokines would contribute to the diagnosis and prediction of the disease development and prognosis.

[Construction of MyD88-Pseudomonas aeruginosa epitope DNA vaccine and its expression in eukaryotic cells].

AIM: To construct the MyD88-Pseudomonas aeruginosa epitope vaccine and study its expression in eukaryotic cells. METHODS: To design and synthesize an epigene containing three B cell epitopes of OprF and one foreign "promiscuous" T cell epitope by overlapping extension PCR. tPA signal encoding sequence was amplified by PCR and then it was inserted into the 5' terminus of the epigene to construct tPA-OprF. tPA-OprF and MyD88 were cloned into the expression vector pIRES and the recombinant plasmid pIRES-tPAOprF-MyD88 was constructed. The recombinant plasmid was transfected into COS-7 cells by electroporation. The expression protein of tPA-OprF and MyD88 was detected by Western blot. RESULTS: The recombinant plasmid pIRES-tPA-OprF-MyD88 was successfully constructed. Western blot analysis indicated the tPA-OprF fusion protein was expressed in supertanant of COS-7 cells and MyD88 protein in COS-7 cells. CONCLUSION: The recombinant plasmid pIRES-tPA-OprF-MyD88 has been successfully constructed and tPA-OprF and MyD88 protein can be highly expressed in transfected cells. It may be used as a potential candidate of preventive vaccine of pseudomonas aeruginosa.

[Cloning and expression of human Runx3 gene obtained from human peripheral blood CD8(+) T lymphocyte].

AIM: Runx3, a type of Runt family member, plays an important role in immune regulation. In this study, we cloned and analyzed the cDNA encoding human Runx3 from T lymphocyte, expressed Runx3 protein in E.coli system, and studied the relation between Runx3 and some immune disorders or tumors. METHODS: The CD8(+) T were isolated from human peripheral blood with MACS, Runx3 cDNA was amplified by RT-PCR and cloned into pMD19-T vector, and recombinant was transformed into competent cells DH5alpha and recombinant sequencing were performed. The identical was subcloned into pQE30 vector and expressed in E.coli M15. The fusion protein was identified by Western blot. RESULTS: The 1,248 bp fragment amplified by RT-PCR was the same as the anticipated one in size and encodes 415 amino acids. Runx3 protein was gained. CONCLUSION: Human Runx3 gene was cloned and expressed in E.coli system successfully, which brought a foundation for further research on its biological function.