Investigating the prognostic and predictive value of the type II cystatin genes in gastric cancer.

BACKGROUND: Accumulating evidence indicates that type II cystatin (CST) genes play a pivotal role in several tumor pathological processes, thereby affecting all stages of tumorigenesis and tumor development. However, the prognostic and predictive value of type II CST genes in GC has not yet been investigated. METHODS: The present study evaluated the expression and prognostic value of type II CST genes in GC by using The Cancer Genome Atlas (TCGA) database and the Kaplan-Meier plotter (KM plotter) online database. The type II CST genes related to the prognosis of GC were then screened out. We then validated the expression and prognostic value of these genes by immunohistochemistry. We also used Database for Annotation, Visualization, and Integrated Discovery (DAVID), Gene Multiple Association Network Integration Algorithm (GeneMANIA), Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), nomogram, genome-wide co-expression analysis, and other bioinformatics tools to analyze the value of type II CST genes in GC and the underlying mechanism. RESULTS: The data from the TCGA database and the KM plotter online database showed that high expression of CST2 and CST4 was associated with the overall survival (OS) of patients with GC. The immunohistochemical expression analysis showed that patients with high expression of CST4 in GC tissues have a shorter OS than those with low expression of CST4 (HR = 1.85,95%CI: 1.13-3.03, P = 0.015). Multivariate Cox regression analysis confirmed that the high expression level of CST4 was an independent prognostic risk factor for OS. CONCLUSIONS: Our findings suggest that CST4 could serve as a tumor marker that affects the prognosis of GC and could be considered as a potential therapeutic target for GC.

Stratifin Promotes Hepatocellular Carcinoma Progression by Modulating the Wnt/ß-Catenin Pathway.

Abnormal stratifin (SFN) expression is closely related to the progression of several human cancers, but the potential roles of SFN in hepatocellular carcinoma (HCC) remain largely unknown. In this study, we found that SFN was upregulated in HCC cell lines and tissues and was positively associated with tumor size, poor differentiation, Tumor Node Metastasis (TNM) stage, and vascular invasion. In addition, high expression levels of SFN were associated with poor overall survival and disease-free survival. Biologically, downregulation of SFN suppressed tumor cell proliferation, epithelial-mesenchymal transition (EMT), invasion, and migration in vitro and tumor growth in vivo. However, overexpression of SFN promoted cell proliferation, EMT, invasion, and migration in vitro and tumor growth in vivo. Mechanistically, overexpression of SFN activated the Wnt/ß-catenin pathway by promoting Glycogen synthase kinase-3 beta (GSK-3ß) phosphorylation, decreasing ß-catenin phosphorylation, promoting ß-catenin transport into the nucleus, and enhancing the expression of c-Myc, whereas depletion of SFN inhibited the Wnt/ß-catenin pathway. In addition, TOPFlash/FOPFlash reporter assays showed that overexpression or downregulation of SFN obviously increased or decreased, respectively, the activity of the Wnt/ß-catenin pathway. Our results indicated that SFN plays an important role in HCC, possibly providing a prognostic factor and therapeutic target for HCC.

ITGA5 Promotes Tumor Progression through the Activation of the FAK/AKT Signaling Pathway in Human Gastric Cancer.

Background: ITGA5 is an adhesion molecule that integrates the intracellular structures with the extracellular matrix to perform biological functions. However, ITGA5 is highly expressed in a variety of tumors and is involved in tumor progression by promoting cell proliferation and metastasis. Nevertheless, little research has been performed on its function in gastric cancer. Therefore, the aim of this study was to investigate the role of ITGA5 in gastric cancer, focusing on the mechanism regulating the proliferation, invasion and migration. Methods: The expression of ITGA5 in gastric cancer tissues was assessed by the use of molecular bioinformatics databases and high-throughput sequencing of gastric cancer tissues from patients. Western blot, qPCR, and immunohistochemistry were performed to detect the expression of ITGA5 in samples from gastric cancer patients and gastric cancer cell lines. Furthermore, the ITGA5 gene was silenced and overexpressed in gastric cancer cells, and the effect on proliferation, invasion, migration, and tumorigenic ability was assessed. Results: ITGA5 mRNA and protein expression were upregulated in gastric cancer cell lines and tissues from patients, and its expression was closely associated with tumor size, lymph node metastasis, and TNM stage. In vitro and in vivo experiments showed that ITGA5 silencing resulted in the inhibition of proliferation, invasion, migration, and graft growth of gastric cancer cells; conversely, the overexpression resulted in the promotion of these cell functions. Our results finally showed that the effect of ITGA5 on proliferation, invasion, and migration of gastric cancer cells was performed through the activation of the FAK/AKT pathway. Conclusions: ITGA5 promotes proliferation, invasion, and migration of gastric cancer cells through the activation of FAK/AKT signaling pathway, suggesting that ITGA5 may be potentially considered as a new target in gastric cancer therapy.

Screening of Serum Exosomal miRNAs as Diagnostic Biomarkers for Gastric Cancer Using Small RNA Sequencing.

Background/Aim: Exosomal miRNAs are promising tumor biomarkers. This research explored the diagnostic value of serum exosomal miRNAs by analyzing the exosomal miRNAs derived from the serum of gastric cancer patients. Methods: Deep sequencing of exosomal miRNAs was performed using an Illumina HiSeq2500 sequencer on serum samples from three healthy subjects in the normal control group (group N) and six gastric cancer patients in the gastric cancer treatment group (group T). Bioinformatics analysis was performed on exosomal miRNA profiles to screen differentially expressed miRNA. In addition, target gene prediction, GO, and KEGG pathway enrichment analyses were performed. Finally, the serum exocrine bodies of 24 patients with gastric cancer and 24 normal controls were verified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to confirm the findings. The receiver operating characteristic (ROC) curve of the subjects was plotted, and the area under the curve (AUC) was calculated with a 95% confidence interval (CI). Results: The exosomes were successfully extracted from the serum of gastric cancer patients, which showed a form of goblet vesicles or irregular circles, with an average particle size of approximately 102.3 nm. The exosomal marker proteins, CD9, CD63, TSG101, and calnexin, were positively expressed. Small RNA sequencing detected 15 different types of RNA components in the serum exosomes, and the most abundant one was miRNA. In the screened cohort, the downregulation of seven existing miRNAs and the upregulation of one existing miRNA were observed. Four of them were selected for confirmation, revealing that the expression of miR-10401-3p, miR-1255b-5p, and miR-6736-5p declined significantly in group T (P < 0.05). In addition, the ROC curve showed that the AUC values for these three miRNAs were 0.8333, 0.8316, and 0.8142, respectively; all of them are statistically significant (P < 0.05). Conclusions: The above three miRNAs found in the serum exosomes from gastric cancer patients might serve as diagnostic biomarkers for gastric cancer.

NKCC1 promotes proliferation, invasion and migration in human gastric cancer cells via activation of the MAPK-JNK/EMT signaling pathway.

Aims: This study aimed to explore the function of NKCC1 in the proliferation, migration and invasion of Gastric cancer (GC) cells. Materials and Methods: GC data extracted from the database was analyzed using molecular bioinformatics. The expression levels of NKCC1 in tissue samples from GC patients and GC cell lines were determined by Western blotting, qRT-PCR, and immunohistochemistry. Immunofluorescence was used to detect protein localization. The GC cell lines were transfected with NKCC1-shRNA or expression plasmid, and in vitro proliferation, invasion and migration were analyzed by the CCK8, wound healing and transwell tests. Results: The NKCC1 mRNA level was significantly increased in GC tissues than that in normal gastric tissues (P = 0.0195). This phenomenon was further confirmed by the analysis of the TCGA-GTEx database that includes 408 gastric cancer tissues and 211 normal gastric tissues (P < 0.01). Furthermore, the increased level of NKCC1 was significantly correlated with Tumor size (P = 0.039), lymphatic node metastasis (P = 0.035) and tumor stage (P = 0.034). In vitro experiments confirmed that NKCC1 expression was higher in GC cells compared to that in GES-1 cells, and was mainly localized to the cytoplasm and membrane. NKCC1 silencing inhibited GC cell proliferation, invasion, migration and EMT, whereas its overexpression had the opposite effects. Furthermore, NKCC1 overexpression upregulated and activated JNK, and the targeted inhibition of JNK by SP600125 abrogated the pro-metastatic effects of NKCC1. Conclusions: NKCC1 promotes migration and invasion of GC cells by MAPK-JNK/EMT pathway and can be a potential therapeutic target.

Expression and Prognostic Analysis of Integrins in Gastric Cancer.

BACKGROUND: Integrins are involved in the biological process of a variety of cancers, but their importance in the diagnosis and prognosis of gastric cancer (GC) is still unclear. Therefore, this study aimed at exploring the significance of ITG gene expression in GC to evaluate its diagnosis and prognosis. METHODS: GEPIA data were used to evaluate the mRNA expression of ITG genes in GC patients. The prognostic value of these genes was assessed by analyzing their mRNA expression using the Kaplan-Meier curve. The biological function of ITG genes was evaluated by GC tissue sequencing combined with GSEA bioinformatics. Based on the sequencing data, ITGA5 with the largest expression difference was selected for verification, and RT-PCR was used to verify its mRNA expression level in 40 pairs of GC and normal tissues. RESULTS: ITG (A2, A3, A4, A5, A6, A11, AE, AL, AM, AV, AX, B1, B2, B4, B5, B6, and B8) was highly expressed in GC tissues, while ITGA8 was low, compared with their expression in normal tissues. RNA-seq data shows that ITG (A2, A5, A11, AV, and B1) expression was associated with poor prognosis and overall survival. In addition, combined with the results of GC tissue mRNA sequencing, it was further found that the differentially expressed genes in the ITGs genes. ITGA5 was highly expressed in GC tissues compared with its expression in normal tissues, as evaluated by qRT-PCR (P < 0.001) and ROC (P < 0.001, AUC (95% CI) = 0.747 (0.641-0.851)), and confirmed that ITGA5 expression was a potential diagnostic marker for GC. Bioinformatics analysis revealed that the signaling pathway involved in ITGA5 was mainly enriched in focal adhesion, ECM-receptor interaction, and PI3K-AKT and was mainly involved in biological processes such as cell adhesion, extracellular matrix, and cell migration. CONCLUSION: This study suggested that ITGs were associated with the diagnosis and prognosis of GC and discovered the prognostic value and biological role of ITGA5 in GC. Thus, ITGA5 might be used as a potential diagnostic marker for GC.

Anticancer Effects of Constituents of Herbs Targeting Osteosarcoma.

Osteosarcoma is a rare primary malignancy of bone that is prone to early metastasis. Resection surgery and chemotherapeutic regimens are current standard treatments for osteosarcoma. However, the long-term survival rate of patients with osteosarcoma is low due to a high risk of metastasis. Hence, a new approach is urgently needed to improve the treatment of osteosarcoma. Compared with chemotherapy, natural active constituents isolated from herbs exhibit less adverse effects and better anti-tumor effects. This study aimed to summarize the anticancer effects of constituents of herbs on the progression and metastasis of osteosarcoma cells. It showed that many constituents of herbs inhibited osteosarcoma by targeting proliferation, matrix metalloproteinases, integrin and cadherin, and angiogenesis. The findings might be beneficial for the development of new drugs and treatment strategies.

PIMT prevents the apoptosis of endothelial cells in response to glycated low density lipoproteins and protective effects of grape seed procyanidin B2.

BACKGROUND: The development of diabetic angiopathy is associated with profound vascular endothelial cells (VEC) dysfunction and apoptosis. Glycated low density lipoproteins (gly-LDL) continuously produced in the setting of diabetic patients play an important role in causing VEC dysfunction and apoptosis. However, the underlying molecular mechanism remains largely elusive. Protein L-isoaspartyl methyltransferase (PIMT) is a widely expressed protein repair enzyme by multiple cell types of arterial wall including VEC. Our previous proteomic studies showed that the expression of PIMT was significantly decreased in the aorta of diabetic rats as compared with control rats and treatment with grape seed procyanidin extracts significantly increased the PIMT expression in diabetic rats. We hypothesized that PIMT plays a critical role in gly-LDL induced VEC apoptosis; grape seed procyanidin B2 (GSPB2) protect against gly-LDL induced VEC apoptosis through PIMT regulation. METHODS AND RESULTS: HUVEC transfected negative control and PIMT siRNA were treated with or without GSPB2 (10 µmol/L) for 48 h. Moreover, HUVEC of PIMT overexpression were stimulated by gly-LDL (50 µg/ml) in the presence or absence of GSPB2 (10 µmol/L) for 48 h. Our results showed that gly-LDL downregulated PIMT expression and PIMT overexpression or GSPB2 significantly attenuated gly-LDL induced VEC apoptosis. PIMT siRNA increased VEC apoptosis with up-regulation of p53, cytochrome c release, caspase-9 and caspase-3 activation. Mechanistically, overexpression of PIMT or GSPB2 increased the phosphorylation of ERK1/2 and GSK3ß in the gly-LDL induced VEC. CONCLUSION: In summary, our study identified PIMT as a key player responsible for gly-LDL induced VEC apoptosis and GSPB2 protect against gly-LDL induced VEC apoptosis by PIMT up-regulation. Targeting PIMT including use of GSPB2 could be turned into clinical application in the fighting against diabetic vascular complications.

Proteomic analysis of kidney and protective effects of grape seed procyanidin B2 in db/db mice indicate MFG-E8 as a key molecule in the development of diabetic nephropathy.

Diabetic nephropathy, as a severe microvascular complication of diabetic mellitus, has become the leading cause of end-stage renal diseases. However, no effective therapeutic strategy has been developed to prevent renal damage progression to end stage renal disease. Hence, the present study evaluated the protective effects of grape seed procyanidin B2 (GSPB2) and explored its molecular targets underlying diabetic nephropathy by a comprehensive quantitative proteomic analysis in db/db mice. Here, we found that oral administration of GSPB2 significantly attenuated the renal dysfunction and pathological changes in db/db mice. Proteome analysis by isobaric tags for relative and absolute quantification (iTRAQ) identified 53 down-regulated and 60 up-regulated proteins after treatment with GSPB2 in db/db mice. Western blot analysis confirmed that milk fat globule EGF-8 (MFG-E8) was significantly up-regulated in diabetic kidney. MFG-E8 silencing by transfection of MFG-E8 shRNA improved renal histological lesions by inhibiting phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), Akt and glycogen synthase kinase-3beta (GSK-3ß) in kidneys of db/db mice. In contrast, over-expression of MFG-E8 by injection of recombinant MFG-E8 resulted in the opposite effects. GSPB2 treatment significantly decreased protein levels of MFG-E8, phospho-ERK1/2, phospho-Akt, and phospho-GSK-3ß in the kidneys of db/db mice. These findings yield insights into the pathogenesis of diabetic nephropathy, revealing MFG-E8 as a new therapeutic target and indicating GSPB2 as a prospective therapy by down-regulation of MFG-E8, along with ERK1/2, Akt and GSK-3ß signaling pathway.

Proteomic analysis of aorta and protective effects of grape seed procyanidin B2 in db/db mice reveal a critical role of milk fat globule epidermal growth factor-8 in diabetic arterial damage.

BACKGROUND: Atherosclerosis is one of the major complications of type 2 diabetic patients (T2DM), leading to morbidity and mortality. Grape seed procyanidin B2 (GSPB2) has demonstrated protective effect against atherosclerosis, which is believed to be, at least in part, a result of its antioxidative effects. The aim of this study is to identify the target protein of GSPB2 responsible for the protective effect against atherosclerosis in patients with DM. METHODS AND RESULTS: GSPB2 (30 mg/kg body weight/day) were administrated to db/db mice for 10 weeks. Proteomics of the aorta extracts by iTRAQ analysis was obtained from db/db mice. The results showed that expression of 557 proteins were either up- or down-regulated in the aorta of diabetic mice. Among those proteins, 139 proteins were normalized by GSPB2 to the levels comparable to those in control mice. Among the proteins regulated by GSPB2, the milk fat globule epidermal growth factor-8 (MFG-E8) was found to be increased in serum level in T2DM patients; the serum level of MFG-E8 was positively correlated with carotid-femoral pulse wave velocity (CF-PWV). Inhibition of MFG-E8 by RNA interference significantly suppressed whereas exogenous recombinant MFG-E8 administration exacerbated atherogenesis the db/db mice. To gain more insights into the mechanism of action of MFG-E8, we investigated the effects of MFG-E8 on the signal pathway involving the extracellular signal-regulated kinase (ERK) and monocyte chemoattractant protein-1 (MCP-1). Treatment with recombinant MFG-E8 led to increased whereas inhibition of MFG-E8 to decreased expression of MCP-1 and phosphorylation of ERK1/2. CONCLUSION: Our data suggests that MFG-E8 plays an important role in atherogenesis in diabetes through both ERK and MCP-1 signaling pathways. GSPB2, a well-studied antioxidant, significantly inhibited the arterial wall changes favoring atherogenesis in db/db mice by down-regulating MFG-E8 expression in aorta and its serum level. Measuring MFG-E8 serum level could be a useful clinical surrogate prognosticating atherogenesis in DM patients.

Grape seed procyanidin b2 inhibits human aortic smooth muscle cell proliferation and migration induced by advanced glycation end products.

Advanced glycation end product (AGE)-induced vascular smooth muscle cell (VSMC) proliferation is vital to the progression of diabetic vasculopathy. A grape seed procyanidin extract has been reported to possess anti-oxidative and anti-inflammatory properties and to display a significant cardiovascular protective effect, but little is know about the underlying mechanism. The objective of this present study was to determine whether GSPB2 (grape seed procyanidin B2), which is a dimeric procyanidin and more biologically active, could inhibit AGE-induced VSMC proliferation by affecting the production of ubiquitin COOH-terminal hydrolase 1 (UCH-L1), the degradation of IκB-α and nuclear translocation of NF-κB in human aortic smooth muscle cells (HASMCs). Our data show that GSPB2 preincubation markedly inhibited AGE-induced proliferation and migration of HASMCs in a dose-dependent manner and upregulated the protein level of UCH-L1. Further studies revealed that the GSPB2 pretreatment markedly attenuated the degradation of IκB-α and nuclear translocation of NF-κB by modulating ubiquitination of IκB-α in AGE-exposed HASMCs. These results collectively suggest that AGE-induced HASMC proliferation and migration was suppressed by GSPB2 through regulating UCH-L1 and ubiquitination of IκB-α. GSPB2 may therefore have therapeutic potential in preventing and treating vascular complications of diabetes mellitus.

Induction of lactadherin mediates the apoptosis of endothelial cells in response to advanced glycation end products and protective effects of grape seed procyanidin B2 and resveratrol.

One of characteristics of diabetes mellitus (DM) is endothelial cell (EC) dysfunction and apoptosis which contributes to the development of vasculopathy. Advanced glycation end products (AGEs) continuously produced in the setting of DM play an important role in causing EC dysfunction and apoptosis. However, the underlying molecular mechanism remains largely elusive. Lactadherin, a secreted glycoprotein of milk-fat globule, is expressed by multiple cell types of arterial wall including ECs. Our previous proteomic studies showed that the expression of lactadherin was significantly increased in the aorta of diabetic rats as compared with control rats and treatment with grape seed procyanidin extracts significantly inhibited the lactadherin expression in diabetic rats. We hypothesized that lactadherin plays a critical role in AGEs-induced EC apoptosis; grape seed procyanidin B2 (GSPB2) and resveratrol protect against AGEs-induced EC apoptosis through lactadherin regulation. Our results showed that AGEs upregulated lactadherin expression and lactadherin RNA interference significantly attenuated AGEs-induced EC apoptosis. Overexpression of lactadherin increased EC apoptosis with up-regulation of Bax/Bcl-2 ratio, cytochrome c release, caspase-9 and caspase-3 activation suggesting the involvement of mitochondria apoptosis pathway. Mechanistically, overexpression of lactadherin reduced the phosphorylation of GSK3beta at baseline. Our study also revealed nine proteins interacting with lactadherin in HUVEC and study of these candidate proteins could unveil further underlying molecular mechanisms. In summary, our study identified lactadherin as a key player responsible for AGEs-induced EC apoptosis and antioxidants GSPB2 and resveratrol protect against AGEs-induced EC apoptosis by inhibiting lactadherin. Targeting lactadherin with antioxidant could be translated into clinical application in the fighting against DM complications.

[Expression and significance of NKG2D ligands in 13 tumor cell lines].

BACKGROUND & OBJECTIVE: The interaction between NKG2D and its ligands plays a major role in immune surveillance against tumor. This study was to observe the expression and analyze the significance of NKG2D ligands in 13 tumor cell lines. METHODS: The mRNA expression of NKG2D ligands in K562, Raji, PG, Hep2, HepG2, HeLa, HT29, M21, MDA231, SGC7901, Caski, HL-60 and Jurkat cells was measured by reverse transcription-polymerase chain reaction (RT-PCR). The cytotoxicity of natural killer (NK) cells to the tumor cells at different effector-to-target cell (E:T) ratios were detected by MTT assay. The expression of MICA protein was measured by SABC immunohistochemistry and Western blot. RESULTS: The 13 tumor cell lines expressed different levels of NKG2D ligands. MICA was highly expressed in Hep2 cells, but not expressed in Caski, PG, HL-60 and Raji cells. The expression of MICA and MICB were positively correlated to the cytotoxicity of NK cells (r=0.851, P<0.001; r=0.652, P<0.05). Except for ULBP3, the expression of ULBP1, 2, 4 had no correlations to the cytotoxicity of NK cells. CONCLUSION: Among the 6 human NKG2D ligands, the expression of MICA is most intimate to the cytotoxicity of NK cells to tumor cells, and its expression level may determine the degree of immune response of NK cells to tumor.

[Multi-central clinical valuation of SHAO's "five needling methods" for treatment of asthma of deficiency of lung and spleen].

OBJECTIVE: To make multi-central clinical valuation of SHAO's "five needling methods" for treatment of asthma of deficiency of lung and spleen. METHODS: Two hundred and ten cases were randomly divided into a test group and a control group, 105 cases in each group. The test group were treated with SHAO's "five needling methods", with Feishu (BL 13), Dazhui (GV 14), Fengmen (BL 12) selected; the control group were treated with routine needling method, with Dingchuan (EX-B 1), Gaohuang (BL 43), Feishu (BL 13), Taiyuan (LU 9), Pishu (BL 20), etc. selected. The treatment in the two groups was given once each day, for 4 weeks, with one-day interval each 6-consecutive day. RESULTS: Fourteen cases were clinically cured, 42 cases were markedly effective, 32 cases were effective and 6 cases were ineffective in the test group with an effective rate of 93.6%; and 8, 30, 41 and 13 cases in the control group, respectively, with an effective rate of 85.9%, with a significant difference between the two groups (P < 0.05). After treatment, the symptoms and signs, pulmonary function in the test group were better than those in the control group (P < 0.05). CONCLUSION: SHAO's "five needling methods" has significant therapeutic effect for treatment of asthma of deficiency of lung and spleen, which is better than that of the routine needling method.

[Up-regulation of IFN-alpha on expression of MICA in the cervical carcinoma cell lines].

AIM: To investigate the effect of IFN-alpha on expression of MHC class I chain-related protein A (MICA) in the cervical carcinoma cell lines. METHODS: The cervical carcinoma cell lines (HeLa and Caski) were treated with IFN-alpha. The expression of MICA was measured by RT-PCR and by immunohistochemical staining. The cytotoxicity of human NK cells to the IFN-alpha treated cervical carcinoma cells was detected by MTT method. RESULTS: The mRNA and protein expression of MICA was up-regulated by IFN-alpha in HeLa and Caski cells in a time and dose-dependent manner. Compared to Caski cells which weakly expressed MICA, higher cytolytic activity of NK cells was found against HeLa cells, which expressed relatively higher level of MICA. After being treated with IFN-alpha for 3 d, the susceptibility of the two cervical carcinoma cells to NK cytolysis was increased significantly. CONCLUSION: IFN-alpha can up-regulate the MICA expression in the cervical carcinoma cell lines and thereby enhance the susceptibility to cytolysis of NK cells.

[Cloning of human NKG2D gene and its expression in CHO cells].

AIM: To construct a recombinant eukaryotic expression vector of human NK cell receptor NKG2D, and express the recombinant human NKG2D in CHO cells. METHODS: A NKG2D gene fragment, with a length of about 650 bp, was amplified from the NK-92 cell line by RT-PCR and was cloned to plasmid pGEM-T Easy. Then the cloned DNA fragment was sequenced. The recombinant plasmid pGEM-T Easy/NKG2D was digested with EcoR I and BamH I, and then NKG2D fragment was isolated and inserted into the corresponding restriction site on eukaryotic expression vector pEGFP-N1. The Lipofectin was used to transfect the recombinant eukaryotic expression plasmid in CHO cells. The expression level of NKG2D gene in transfected CHO cells was detected by fluorescence microscope, RT-PCR, Western blot and immunohistochemical staining. RESULTS: The length of cDNA fragment amplified by RT-PCR was consistent with that of NKG2D. DNA sequencing of pGEM-T Easy/NKG2D revealed that the cloned DNA sequence was identical to that of reported NKG2D. Green fluorescence was seen in transfected CHO cells by fluorescence microscope. Human NKG2D mRNA was highly expressed in transfected CHO cells. Western blot and Immunohistochemical staining detection showed that NKG2D was expressed in transfected cells. CONCLUSION: A recombinant eukaryotic expression vector of human NKG2D can be constructed and it can be expressed successfully in CHO cells.

[Negative Regulatory Effect of T-Lymphocytes on the Expansion of Human Bone Marrow Hematopoietic Cells In Vitro]

In this research, the regulatory effects of T-lymphocytes on the expansion of hematopoietic cells in human bone marrow were studied. Anti-CD3 McAb and anti-CD8 McAb were used to eliminate the T-lymphocytes in bone marrow MNCs. Cell surface antigens were analysed by flow cytometry. Hematopoietic cells expanded with hematopoietic growth factors (HGFs) in a liquid culture system and the number of CD34(+) cell, CFU-GM and CFU-GEMM were determined. After cultured with HGFs for 20 days the total number of cells expanded by 75.8, 79.6, 77.4 and 67.0 folds respectively in three experimental groups (anti-CD3 McAb, anti-CD8 McAb and anti-CD3 + anti-CD8 McAb) and control group (MNC of bone marrow). The number of CFU-GM in the four groups were 173.67 +/- 18.90, 165.33 +/- 26.58, 170.33 +/- 21.50 and 79.67 +/- 8.33 respectively. The number of CFU-GEMM in the four groups were 431.33 +/- 34.56, 370.33 +/- 42.10, 386.67 +/- 10.02 and 177.67 +/- 26.86 respectively. There were significant differences in the number of CFU-GM and CFU-GEMM between experimental groups and control group. The results showed that the T-lymphocytes in bone marrow could inhibit the expansion of hematopoietic cells in vitro and the formation of CFU-GM and CFU-GEMM. The regulatory mechanism was to be explored.