Altered intestinal microbiota in children with bronchiolitis.

Objective: To investigate the correlation between the alteration of intestinal microbiota and disease in children with bronchiolitis. Methods: Fifty seven children diagnosed with bronchiolitis from January 2020 to January 2022 in our pediatric department were included as the case group, and another 36 normal children were included as the control group. Stool and blood were collected from both groups for high-throughput sequencing, untargeted metabolite detection and ELISA. A mouse model of RSV infection was established to validate the results of clinical case detection. Results: Body weight, passive smoking, and a host of other factors were possible as acute bronchiolitis influencing factors in the onset of acute bronchiolitis. The alpha diversity Shannon, Simpson and Pielou's evenness indices were significantly lower in children with acute bronchiolitis than in healthy children with gated levels of Firmicutes, Bacteroidetes and genus levels of Clostridium and other short chain fatty acid-producing bacteria. The relative abundance of short-chain fatty acid (SCFAs)-producing bacteria decreased and the abundance of genus-level sphingolipid-producing bacteria Sphingomonas increased; the progression of acute bronchiolitis is likely to be associated with the abundance of Clostridium and Sphingomonas and higher fecal amino acid concentrations, including FF-MAS, L-aspartic acid, thioinosinic acid, picolinic acid; supplementation with Clostridium butyricum significantly alleviated RSV infection-induced lung inflammation. Conclusion: The progression of bronchiolitis may be associated with altered intestinal microbiota, decreased SCFAs and elevated sphingolipids metabolism in children. Some fecal bacteria and metabolites may predict the onset of bronchiolitis, and oral administration of Clostridium butyricum may alleviate RSV infection-induced pulmonary inflammation.

SGLT2 inhibitors attenuate nephrin loss and enhance TGF-ß1 secretion in type 2 diabetes patients with albuminuria: a randomized clinical trial.

To evaluate the effect of SGLT2 inhibitor (SGLT2i) on albuminuria, nephrin (NPH) and transforming-growth-factor-beta1 (TGF-ß1) levels in urine and low-grade inflammation in type 2 diabetes (T2D) patients. A randomized, blank-controlled clinical trial included 68 T2D patients and 10 controls. Based on the urinary albumin-to-creatinine ratio (UACR), 68 diabetic patients were stratified into three levels, UACR < 30 mg/g, UACR ≧ 30 mg/g to ≦ 300 mg/g and UACR ˃ 300 mg/g, who were randomized (1:1:1) to receive SGLT2i treatment for 12 weeks. The concentrations of NPH and TGF-ß1 in urine were measured as indications of podocyte injury and renal fibrosis. Low-grade inflammation was assessed by the levels of IL-6, TNFα and hsCRP. After 12 weeks of SGLT2i treatment, the levels of UACR and NPH decreased, UTGF-ß1 increased in the T2D with microalbuminuria and macroalbuminuria groups, NPH (1.12 [0.59, 1.29] vs. 0.71 [0.41, 1.07] µg/ml, P = 0.022) and (1.29 [0.99, 1.96] vs. 0.93 [0.57, 1.31] µg/ml, P = 0.002), UTGF-ß1 (4.88 ± 1.31 vs. 7.27 ± 1.21 pg/ml, P  < 0.001) and (4.30 ± 1.34 vs. 6.78 ± 2.59 pg/ml, P  < 0.001), respectively. The changes in NPH were positively correlated with the UACR and negatively correlated with UTGF-ß1 in T2D with albuminuria. SGLT2i alleviate nephrin loss and enhance TGF-ß1 excretion in urine in T2DM with albuminuria. The anti-albuminuric effect of SGLT2i could be attributed to mitigating podocyte apoptosis and attenuating renal fibrosis.Trial registration This clinical trial was registered on 15/10/2019, in ClinicalTrials.gov, and the registry number is NCT04127084.

Multidisciplinary team therapy for left giant adrenocortical carcinoma: A case report.

BACKGROUND: Adrenocortical carcinoma (ACC) is a rare malignant epithelial tumor originating from adrenocortical cells that carries a very poor prognosis. Metastatic or inoperable diseases are often considered incurable, and treatment remains a challenge. Especially for advanced cases such as ACC complicated with renal venous cancer thrombus, there are few cumulative cases in the literature. CASE SUMMARY: The patient in this case was a 39-year-old middle-aged male who was admitted to the hospital for more than half a month due to dizziness and chest tightness. Computed tomography (CT) findings after admission revealed a left retroperitoneal malignant space-occupying lesion, but the origin of the formation of the left renal vein cancer thrombus remained to be determined. It was speculated that it originated from the left adrenal gland, perhaps a retroperitoneal source, and left adrenal mass + left nephrectomy + left renal vein tumor thrombus removal + angioplasty were performed under general anesthesia. Postoperative pathology results indicated a diagnosis of ACC. Postoperative steroid therapy was administered. At 3 mo after surgery, abdominal CT reexamination revealed multiple enlarged retroperitoneal lymph nodes and multiple low-density shadows in the liver, and palliative radiotherapy and mitotane were administered, considering the possibility of metastasis. The patient is currently being followed up. CONCLUSION: ACC is a highly malignant tumor. Even if the tumor is removed surgically, there is still the possibility of recurrence. Postoperative mitotane and adjuvant chemoradiotherapy have certain benefits for patients, but they cannot fully offset the poor prognosis of this disease.

Identification of the potential novel biomarkers as susceptibility gene for Wilms tumor.

BACKGROUND: Wilms tumor (WT) is the most common malignant renal tumor in children. The aim of this study was to identify potential susceptibility gene of WT for better prognosis. METHODS: Weighted gene coexpression network analysis is used for the detection of clinically important biomarkers associated with WT. RESULTS: In the study, 59 tissue samples from National Cancer Institute were pretreated for constructing gene co-expression network, while 224 samples also downloaded from National Cancer Institute were used for hub gene validation and module preservation analysis. Three modules were found to be highly correlated with WT, and 44 top hub genes were identified in these key modules eventually. In addition, both the module preservation analysis and gene validation showed ideal results based on other dataset with 224 samples. Meanwhile, Functional enrichment analysis showed that genes in module were enriched to sister chromatid cohesion, cell cycle, oocyte meiosis. CONCLUSION: In summary, we established a gene co-expression network to identify 44 hub genes are closely to recurrence and staging of WT, and 6 of these hub genes was closely related to the poor prognosis of patients. Our findings revealed that those hub genes may be used as potential susceptibility gene for clinical diagnosis and prognosis of this tumor.

Oridonin induces apoptosis in HGC-27 cells by activating the JNK signaling pathway.

Gastric cancer (GC) is a very common type of cancer. Although current treatment modalities include surgical resection and chemotherapy, many patients are either not eligible for radical resection or have a poor response to chemotherapy. Due to the complex features of the disease, there is a need for complementary therapy. In the present study, the effects of oridonin on cell proliferation, invasion and apoptosis were assessed in the HGC-27 cell line using the Cell Counting Kit-8 assay, real-time cell analysis, and an Annexin V-FITC/propidium iodide (PI) detection kit, respectively. The effect of oridonin on apoptosis, through the JNK pathway, was also investigated using western blotting. The present study demonstrated that oridonin can suppress cell viability and inhibit cell proliferation by inducing G2/M arrest. Oridonin also induced caspase-dependent apoptosis in cells by activating the phosphorylated-JNK/C-JUN pathway. These results demonstrate the potential of oridonin as a potential therapeutic compound for the treatment of GC.

KRAS G12V Mutation is an Adverse Prognostic Factor of Chinese Gastric Cancer Patients.

This study aims to investigate the molecular characteristics of Chinese gastric cancer patients. In our study, the KRAS, BRAF, and PIK3CA mutation status of 485 GC patients were analyzed by Sanger sequencing. Kaplan-Meier analysis was used to plot survival curves according to different genotypes. The results show that the frequency of KRAS, BRAF and PIK3CA mutations were 4.1%, 1.2% and 3.5%, respectively. BRAF mutations were significantly concentrated in stage III and IV gastric cancer (P=0.009). KRAS G12V mutation carriers have much shorter OS than other mutation carriers and wild-type group patients (P=0.013). In conclusion, only the KRAS G12V mutation has an adverse effect on patient survival.

Prognostic Significance of FKBP14 in Gastric Cancer.

INTRODUCTION: Although our understanding on gastric cancer biology is better than a decade ago, its practical effect on screening and diagnosis remains limited. Moreover, there are no markers that can be accurately used in the clinic to diagnose early-stage gastric cancer or monitor the patient's response to therapy. Herein, we investigate whether FKBP14 is involved in the progression of gastric cancer. METHODS: The AGS cell line was chosen for over-expression analysis, whereas the SGC-7901 cell line was selected for knock-down analysis. AGS cells were transfected with an FKBP14 overexpression plasmid (AGS-PLV.O-FLAG). The expression pattern of FKBP14 in both cell lines was determined by Western blot and RT-PCR. Cell proliferation was assessed using Cell Counting Kit-8, whereas apoptosis was performed using flow cytometry. The expression of FKBP14 in 70 Chinese patients with gastric cancer was also investigated using tissue microarrays and compared with gastric cancer patients from The Cancer Genome Atlas. RESULTS: FKBP14 was highly expressed in SGC7901 and had a relatively low expression in AGS cells. Upregulation of FKBP14 in AGS cells promoted migration and invasion and inhibits apoptosis. Knock-down of FKBP14 resulted in a suppression in migration and invasion and promoted apoptosis in the SGC-7901 cell line. Effectively, gastric cancer patients had a higher expression of FKBP14, with a lower survival rate (P = 0.028). Patients with a high expression of FKBP14 were significantly correlated with lymph node metastasis (P =0.016), and an advanced histologic grade (P =0.021). CONCLUSION: FKBP14 is often up-regulated in gastric cancer. Patients with a high expression of FKBP14 are usually associated with worse overall survival. FKBP14 is an oncogene in gastric cancer, and is a potential biomarker for GC diagnosis, invasion, and prognosis.

A value-added approach to improve the nutritional quality of soybean meal byproduct: Enhancing its antioxidant activity through fermentation by Bacillus amyloliquefaciens SWJS22.

The action of ß-glucosidase and protease of Bacillus amyloliquefaciens SWJS22 in the fermentation of soybean meal caused a significant increase 1): in total phenolic and flavonoid contents with two-fold or higher, largely associated with daidzein, glycitein, genistein, protocatechuic, and p-hydroxybenzoic, gallic acids; 2): the amount of peptides <3 kDa, maillard reaction intermediate and maillard reaction product with five-, three- and twenty-one-fold, respectively. The significant increase in the amount of antioxidant components in the lyophilised fermented soybean meal supernatant (LFSMS) was associated with the improved antioxidant activity. Namely, the DPPH radical scavenging activity, reducing power, and oxygen radical absorbance capacity of the LFSMS generally increased, and LFSMS (at doses >250 mg/kg body weight) improved the activities of superoxide dismutase, glutathione peroxidase, catalase and total antioxidant capacity, and inhibited the formation of malondialdehyde in mouse serum and liver (p < 0.05). Therefore, LFSMS could be used as functional food ingredients.

Heterochromatin protein HP1γ promotes colorectal cancer progression and is regulated by miR-30a.

Colorectal cancer pathogenesis remains incompletely understood. Here, we report that the heterochromatin protein HP1γ is upregulated commonly in human colorectal cancer, where it promotes cell proliferation in vitro and in vivo. Gene-expression and promoter-binding experiments demonstrated that HP1γ directly regulated CDKN1A (p21(Waf1/Cip1)) in a manner associated with methylation of histone H3K9 on its promoter. We identified miR-30a as a tumor-suppressive microRNA that targets HP1γ in vitro and in vivo to specifically suppress the growth of colorectal cancer in mouse xenograft models. MiR-30a was widely downregulated in primary human colorectal cancer tissues, where its expression correlated inversely with high levels of HP1γ protein. Our results identify a new miR-30a/HP1γ/p21 regulatory axis controlling colorectal cancer development, which may offer prognostic and therapeutic opportunities.

LYAR promotes colorectal cancer cell mobility by activating galectin-1 expression.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. However, the molecular mechanisms of CRC pathogenesis are not fully understood. In this study, we report the characterization of LYAR (Ly-1 antibody reactive clone) as a key regulator of the migration and invasion of human CRC cells. Immunohistochemistry analysis demonstrated that LYAR is expressed at a higher level in metastatic CRC tissues. We found that LYAR promoted the migratory and invasive capabilities of CRC cells. Gene expression profile analysis of CRC cells showed that LGALS1, which encodes the galectin-1 protein, was a potential target of LYAR. The ChIP assay and gene reporter assays indicated that LYAR directly bound to the LGALS1 promoter. The ectopic expression of galectin-1 partially restored the mobile potential of LYAR knocked-down cells, which suggests that galectin-1 contributed to the LYAR-promoted cell migration and invasion of CRC cells. Thus, this study revealed a novel mechanism by which the transcription factor LYAR may promote tumor cell migration and invasion by upregulating galectin-1 gene expression in CRC.

Pancreatic Insulinoma Misdiagnosed as Epilepsy for Eight Years: A Case Report and Literature Review.

A 58-year-old Chinese man presented with intermittent seizure episodes after being misdiagnosed with epilepsy for eight years. MRI revealed an abnormally strong signal in the distal pancreas. The patient was subsequently diagnosed with pancreatic insulinoma based on the histological findings, and his symptoms improved following surgical removal of the tumor. The appearance of unusual manifestations of insulinoma makes it difficult to diagnose the condition. This disorder should be included in the differential diagnosis of epilepsy and mental illness.

The clinical significance and risk factors of solitary lymph node metastasis in gastric cancer.

AIMS: To assess the clinical significance and risk factors of solitary lymph node metastasis (SLM) in gastric carcinoma and establish a more accurate method to evaluate the possibility of lymph node metastasis (LM). METHODS: A total of 385 patients with gastric carcinoma who underwent D2 lymphadenectomy at the Cancer Center of Sun Yat-Sen University were included in this research. Then we used a group of data from Sun Yat-sen University Gastrointestinal Hospital (SYSUGIH) to validate the accuracy of our developed method. The χ2 test, Kaplan-Meier analysis, log-rank test, COX model, and discriminate analysis were used to analyze the data with SPSS13.0. RESULTS: We found that the LM number and pathological T staging were independent prognostic risk factors. CEA grading, LN status by CT, and T staging by CT were independent risk factors for LM in gastric carcinoma. In addition, we developed the equation Y = -5.0 + X1 + 1.8X3 + 0.7X4 (X1 = CEA grading, X3 = LN status by CT, X4 = T staging by CT) to evaluate the situation of LM. The data from SYSUGIH shows this equation has a better accuracy compared with CT. CONCLUSIONS: SLM is an independent risk factor in gastric cancer. And there was no survival difference between the skip metastasis group and the other SLM group (P = 0.659). It is inappropriate for the patient with SLM doing a standard D2 lymphadenectomy, due to the fact that LM rarely occurs in the splenic artery, splenic hilum. The risk factors for LM include CEA grading, LN status by CT, and T staging by CT. And we can use Y = -5.0 + X1 + 1.8X3 + 0.7X4 (X1, CEA grading, X3 = LN status by CT, X4 = T staging by CT, the critical value is 0.3) to estimate the possibility of LM, which has a better accuracy compared with CT.

Enteral supplementation of alanyl-glutamine attenuates the up-regulation of beta-defensin-2 protein in lung injury induced by intestinal ischemia reperfusion in rats.

BACKGROUND: Beta-defensin-2 (BD-2), an endogenous antimicrobial peptide, plays a key role in immune response against microbial invasion. This study aimed to observe the effect of Alanyl-Glutamine (Ala-Gln) on BD-2 protein expression in pulmonary tissues after intestinal ischemia reperfusion (IIR) in rats and to investigate its correlations to pulmonary inflammatory and oxidative injury. METHODS: Rats in IIR and the two treatment groups were subjected to intestine ischemia for 60 min and those in the treatment groups were administered orally with Ala-Gln or alanine (Ala) respectively. Lung tissues were harvested to detect the BD-2 protein expression. Concentrations of Tumor necrosis factor (TNF)-α and malondialdehyde (MDA) as well as superoxide dismutase (SOD) activity in lung tissues were determined simultaneously. RESULTS: Ala-Gln attenuated the up-regulation of BD-2 expression (p < 0.05) and TNF-α (p < 0.05), MDA (p < 0.05) levels, as well as the reduction of SOD activity (p < 0.05) in lung tissues after IIR. But Ala did not exert significant effects. BD-2 protein in lung tissues was positively correlated to local TNF-α level (p < 0.01) and MDA concentration (p < 0.01) with statistical significance. CONCLUSION: Ala-Gln can relieve the IIR-induced up-regulation of BD-2 protein expression in the lung of rats, which involves anti-inflammation and anti-oxidation mechanisms.

[Bone marrow-derived dendritic cells rather than spleen-derived dendritic cells can generate regulatory T cells].

There are many evidences that dendritic cells (DC) can establish and maintain immunological tolerance through inducing the differentiation of regulatory T cells Treg. This study was purposed to explore the possibility to gene-rate Treg from bone marrow-derived DC (BM-DC) or spleen-derived DC (spDC) generated CD4(+) CD25(+) FOXP3(+) Treg by induction. Bone marrow immature DC (imDC) induced from bone marrow precursor cells of C57BL/6 mice by GM-CSF and IL-4; after culture for 6 day, imDC were stimulated by LPS for additional 16 hours and the mature DC (mDC) have been got; the spDC were collected from spleen of C57BL/6 mice by MACS. Co-culturing fresh BALB/c mouse CD4(+) T cells with these three sorts of DC above mentioned respectively was performed to generate CD4(+) CD25(+) FOXP3(+) Treg. The expression of FOXP3 in CD4(+) T cells was detected by flow cytometry, and the capacity of different DC generated CD4(+) CD25(+) FOXP3(+) Treg was evaluated. The results showed that stimulated by C57BL/6 immature or mature DC, the positive rate of FOXP3 in BALB/c CD4(+) T cells increased from (8.57 ± 1.14)% to (15.80 ± 1.35)%, (17.93 ± 1.45)% respectively (P < 0.01); while stimulated by spDC, the positive rate of FOXP3 in BALB/c CD4(+) T cells decreased from (8.57 ± 1.14)% to (3.95 ± 0.79)% (P < 0.05). It is concluded that the BM-DC but not spDC can generate Treg from CD4(+) T cells, BM-DC may mediate immune tolerance rather than the immune response.

MCPH1 Protein Expression in Normal and Neoplastic Lung Tissues.

Lung cancer is the most common cause of cancer-related death in the world. The main types are small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC), the latter including squamous cell carcinoma (SCC), adenocarcinoma and large cell carcinoma. NSCLCs account for about 80% of all lung cancer cases. Microcephalin (MCPH1), also called BRIT1 (BRCT-repeat inhibitor of hTERT expression), plays an important role in the maintenance of genomic stability. Recently, several studies have provided evidence that the expression of MCPH1 gene is decreased in several different types of human cancers. We evaluated the expression of protein MCPH1 in 188 lung cancer and 20 normal lung tissues by immunohistochemistry. Positive MCPH1 staining was found in all normal lung samples and only some cancerous tissues. MCPH1-positive cells were significantly lower in lung carcinoma compared with normal tissues. Furthermore, we firstly found that MCPH1 expression in lung adenocarcinoma is higher than its expression in squamous cell carcinoma. Change in MCPH1 protein expression may be associated with lung tumorigenesis and may be a useful biomarker for identification of pathological types of lung cancer.

BAT-25 polymorphism in Chinese from Jiangsu province and its implication for locus microsatellite instability screening.

BACKGROUND: Colorectal cancer is one of the most common tumors with high mortality in China. Microsatellite instability (MSI) analysis is important for the diagnosis of hereditary non-polyposis colorectal cancer (HNPCC) and for the prediction of 5-FU chemotherapy efficiency of colorectal tumors, especially in terms of therapeutic response and overall survival rates. Among the MSI markers recommended by the NIH/NCI, BAT-25 has been extensively studied for its major role in MSI. BAT-25 presents different polymorphisms in different ethnic populations and studies of its polymorphisms in the Chinese population are still very limited. AIMS: To analyze the frequency of constitutive polymorphic variation at the BAT-25 locus in Chinese from Jiangsu Province and its implication for locus MSI screening. METHODS: The frequency of allelic variation at the BAT-25 locus of cervical cells from 500 healthy women and blood from 16 healthy males was assessed by direct sequencing. Twenty samples were also analyzed by fragment analysis. DNA extracted from blood of 94 patients with gastrointestinal cancer or endometrial cancer was analyzed by fragment analysis. RESULTS: After comparison with the sequencing results, the more frequent allele lengths were 126-127 bp, 128-129 bp, 129-130 bp, respectively consistent with the 24 poly(T) (T24), T25 and T26 alleles. At the BAT-25 locus, 516 healthy individuals had respectively 1.36%, 97.28% and 1.36% of the T24, T25 and T26. Whereas for the 94 cancer patients allelic frequencies were 0.53%, 1.06%, 96.8%, 1.6% for T15, T24, T25 and T26 alleles respectively. Sixteen healthy males had only the T25 allele and heterozygous T15 was only found in 1 male patient with colon cancer. CONCLUSION: We established the relation between fragment length and thymine repeats in BAT-25. The results showed that the BAT-25 locus is quasimonomorphic in Chinese from Jiangsu province. Moreover we showed that variant alleles of BAT-25 were found more likely in blood from cancer patients than in healthy individuals, suggesting the need to perform comparative studies between tumor and blood, or normal tissue, as to obtain a correct MSI identification.

Tissue metabolomic fingerprinting reveals metabolic disorders associated with human gastric cancer morbidity.

The principal way to improve the outcome of gastric cancer (GC) is to predict carcinogenesis and metastasis at an early stage. The aims of the present study were to test the hypothesis that distinct metabolic profiles are reflected in GC tissues and to further explore potential biomarkers for GC diagnosis. Gas chromatography/mass spectrometry (GC/MS) was utilized to analyze tissue metabolites from 30 GC patients. A diagnostic model for GC was constructed using orthogonal partial least squares discriminant analysis (OPLS-DA), and the metabolomic data were analyzed using the non-parametric Wilcoxon rank sum test to identify the metabolic tissue biomarkers for GC. Over 100 signals were routinely detected in one single total ion current (TIC) chromatogram, and the OPLS-DA model generated from the metabolic profile of the tissues adequately discriminated the GC tissues from the normal mucosae. Among the low-molecular-weight endogenous metabolites, a total of 41 compounds, such as amino acids, organic acids, carbohydrates, fatty acids and steroids, were detected, and 15 differential metabolites were identified with significant difference (p<0.05). A total of 20 variables were noted which contributed to a great extent in the discriminating OPLS-DA model (VIP value >1.0), among which 12 metabolites were identified using both VIP values (VIP >1) and the Wilcoxon test (p<0.05). In conclusion, the identification of the metabolites associated with GC morbidity potentially revealed perturbations of glycolysis, fatty acid ß-oxidation, cholesterol and amino acid metabolism. These results suggest that tissue metabolic profiles have great potential in detecting GC and may aid in understanding its underlying mechanisms.

[Systematic review on safety and efficacy of laparoscopy-assisted distal gastrectomy with D2 lymph node dissection for gastric cancer].

OBJECTIVE: To evaluate the safety and efficacy of laparoscopy- assisted distal gastrectomy (LADG) with D2 lymph node dissection for gastric cancer. METHODS: Literature search was performed in Pubmed, Medline, EMBASE, the Chinese Biomedical Database (CBM) to identify controlled trials comparing LADG and open distal gastrectomy (ODG) for gastric cancer published between January 2005 and February 2010. A meta-analysis was performed using RevMan 5.0 software. RESULTS: Seven controlled trials were included. One trail was randomized controlled trial. Compared to ODG, LADG had less blood loss[WMD: -132.04, 95% confidence interval (CI): -207.32 to -56.77], earlier postoperative first flatus (WMD: -0.82, 95% CI: -1.20 to -0.45], less complications [odds ratio (OR): 0.45, 95% CI: 0.26 to 0.78], shorter postoperative hospital stay (WMD: -3.63, 95% CI: -4.19 to -3.07), more harvested lymph nodes (WMD: 1.93, 95%CI: 0.36 to 3.50). There were no significant differences between the two groups in recurrence rate, metastasis rate, mortality and survival rate. CONCLUSION: Short-term outcome of LADG with D2 lymph node dissection for gastric cancer is superior to ODG.

[Transfection efficiency of adenoviral vector AD5/F35 to malignant hematopoietic cells of different origins].

This study was aimed to investigate the transfection efficiency of adenoviral vector AD5/F35 to hematopoietic malignant cells lines of various origins and AD5/F35 cytotoxicity. The hematologic malignant cell lines of various origins were transfected by AD5/F35-EGFP at different multiple of infection (MOI) and AD5-EGFP was used as control; the proportion of fluorescence positive cells was detected by flow cytometry; the killing effect of virus on infective target cells was assayed by MTT and observed by fluorescence microscopy. The results showed that the transfection efficiency of AD5/F35 vector to cell line of myeloid origin was > 99% at MOI = 30, the transfective efficiency of AD5 vector was 26.4% at MOI = 1,000; the transfection efficiency of AD5/F35 vector and AD5 vector to cell line of B cell origin were 11.7% and 5.7%, respectively, at MOI = 1,000. AD5/F35 and AD5 vectors could not effectively transfect cells of T cell origin, no fluorescence positive cells were detected at MOI = 1,000; no significant killing effect of AD5/F35 vector on infective target cells was observed at MOI = 1,000. It is concluded that AD5/F35 vector infection has definite selectivity to hematologic malignant cells of various origin, the infection ability of AD5/F35 vector to cells of myeloid origin is stronger than that to cells of B cell origin, the cytotoxicity of AD5/F35 vector to infective target cells is small. The AD5/F35 vector is preferable to AD5 vector in respect of infection ability and offers good prospects of application in gene therapy for myeloid leukemia cells as target cells.

[Construction of rAAV2-GPIIb/IIIa vector and test of its expression and function in vitro].

This study was aimed to explore the possibility of rAAV2 vector-mediating gene therapy for Glanzmann' s thrombasthenia. The rAAV2-GPIIb/IIIa vector was constructed. The GPIIb/IIIa gene expression in mammal cell were examined by different methods, such as: detection of mRNA expression in BHK-21 cells after 24 hours of infection (MOI = 1 x 10(5) v.g/cell) was performed by RT-PCR; the relation between MOI and quantity of GPII6/IIIa gene expression was detected by FACS after 48 hours of infection; GPIIb/IIIa protein expression in BHK-21 cells after 48 hours of infection (MOI = 10(5) v x g/cell) was assayed by Western blot, GPIIb/IIIa protein expression on cell surface was detected by immunofluorescence, and the biological function of expressing product was determined by PAC-1 conjunct experiments. The results showed that GPIIb/IIIa gene expression in mRNA level could be detected in BHK-21 cells after 24 hours of infection at MOI = 1 x 10(5) v x g/cell and the GPIIb/IIIa gene expression in protein level could be detected in BHK-21 cells after 48 hours of infection at MOI = 1 x 10(5) v x g/cell. In certain range, quantity of GPIIb/IIIa gene expression increased with MOI, but overdose of MOI decreased quantity of GPIIb/IIIa gene expression. Activated product of GPIIb/IIIa gene expression could combined with PAC-I, and possesed normal biological function. In conclusion, rAAV2 vactor can effectively mediate GPIIb and GPIIIa gene expressing in mammal cells, and the products of these genes exhibit biological function. This result may provide a basis for application of rAAV2 vector in Glanzmann's thrombasthenia gene therapy in furture.