BACKGROUND: Studies of biological characteristics of mesenchymal stem cells (MSCs) and regulatory factors that influenced the differentiation of MSCs have shown that the proportion of the natural differentiation from in vitro primarily cultured MSCs into hepatocytes was low, and to select a suitable inductor is important to enhance the differentiation of MSCs into hepatocytes.OBJECTIVE: To verify the feasibility of induced differentiation of rat bone marrow MSCs (BMSCs) into hepatocytes using the combination of hepatocyte growth factor (HGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF-4).DESIGN, TIME AND SETTING: The cytological in vitro study was performed at the Experimental Center, Weifang Medical College in August 2007.MATERIALS: Totally 40 Sprague-Dawley rats were supplied by the Experimental Animal Center, Weifang Medical College.METHODS: Rat BMSCs were incubated by adherent method. BMSCs at passage 3 were assigned to 2 groups. BMSCs in the blank control group were treated with L-DMEM containing 10% fetal bovine serum. BMSCs in the combination group were treated with 10 μg/L FGF, 8 μg/L HGF and 8 μg/L EGF following above-mentioned procedures.MAIN OUTCOME MEASURES: Inverted microscope was used to observe the morphological changes in cells.Immunofluorescence method was used to observe the expression of alpha-fetoprotein (AFP) and albumin (ALB). PAS was employed to detect the expression of glycogen. Fox green intake experiment was conducted. Enzymology was utilized to test the contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP).RESULTS: BMSCs in the combination group presented polygonal, orbicular or round shape. BMSCs in the blank control group remained spindle. BMSCs in the combination group were positive for AFP and ALB at day 14 following culture, and a few PAS-positive and fox green-positive cells were found at day 7. Positive cells became more over time. Synthesis of ALT, AST and ALP was detected at day 14, reached a peak at 21 days, and then decreased. Above-described indexes were negative in the blank control group.CONCLUSION: After induced by the FGF, HGF and EGF, BMSCs have the ability to differentiate into hepatocytes in vitro.

Objective To explore the possibility that rat bone mesenchymal cells (BMSCs) can differentiate into hepatocytes under the affection of fetal liver filtrate. Methods PAS and green indigo dye were used to detect glycogen and differential level of hepatocytes, respectively. The concentration of ALT, AST, ALP in the culture supernatant were served as markers of hepaocyte function. Results Fourteen days after induced by the fetal liver filtrate, BMSCs changed their shapes into polygon, oval or round. Some of BMSCs were positive for AFP and ALB at 7 days after induction, then the number of positive cells increased, and most of BMSCs expressed AFP and ALB till 21days. The PAS reaction and indocyanine green(ICG) intaking also appeared at 7days. Enzyme in supernatant such as ALT, AST, ALP were fristly detected at 7days and peaked at 14days,then the level declined. Conclusion The fetal rat liver filtrate was able to induce BMSCs into cells with function and characteristics of hepatocytes.

Objective To construct microRNA (miRNA) expression vector targeting surviving,and to investigate its effect on transfected human colorectal carcinoma (HT-29) cell apoptosis and proliferation.Methods miRNA targeting survivin was synthesized and transfected HT-29 cells by lipofectin.HT-29 cells were cultured in the 6 orifices.The cultured cells were divided into control,liposome,negative control and positive control groups.Transient transfected cells were collected and the proliferation index and apoptosis rate of HT-29 cells were detected by flow cytometry.The expressions of survivin mRNA and protein were detected by RT-PCR and Western blot.Results The proliferation index and apoptosis rate of the positive control group were significantly higher compared with normal group,transfection group and mock-vehicle group (17.98％ ± 2.35％ vs 38.04％ ±2.11％ vs 36.73％ ±2.51％ vs 36.57％ ±3.05％; t =20.05,P＜0.01; t =18.75,P＜0.01; t=18.59,P＜0.01; 19.54％ ±1.74％ vs 3.13％ ±0.29％ vs 3.70％ ±0.44％ vs 3.61％ ± 0.50％ ; t =16.40,P ＜ 0.01 ; t =15.84,P ＜ 0.01 ; t =15.92,P ＜ 0.01).Survivin mRNA and protein expression levels were specifically suppressed in transfected HT-29 cells (t =0.68,P ＜0.01 ; t =0.58,P ＜ 0.01; t=0.61,P＜0.01;t=0.64,P＜0.01; t=0.62,P＜0.01;t=0.67,P＜0.01).Conclusion Survivin targeted silence can effectively decrease the expression of survivin mRNA and protein,induce colorectal carcinoma HT-29 cell apoptosis and inhibit cell proliferation.

Objective: To investigate the expression of ATP-Binding Cassette Proteins including P-gp (P-glycoprotein), MRP1 (multidrug resistance associated protein 1) and BCRP (breast cancer resistance protein) in hepatocellular carcinoma and its relationship with pathological features. Methods: The expression of P-gp/MDR1 (multidrug resistance gene 1), MRP1 and BCRP in hepatocellular carcinoma was examined by RT-PCR and immunohistochemistry in 34 cases of hepatocellular carcinoma and 19 cases of paraneoplastic hepatic tissues. Results: The expression of MDR1, MRP1 and BCRP mRNA (messenger ribonucleic acid) was 1.15±0.24, 0.64±0.33, and 1.07±0.32 in hepatocellular carcinoma and 0.36±0.14, 0.19±0.06, and 0.31±0.09 in paraneoplastic hepatic tissues. The expression of MDR1, MRP1 and BCRP mRNA was 1.38±0.26, 0.73±0.35, and 1.34±0.21 in poorly differentiated hepatocellular carcinoma and 0.74±0.32, 0.30±0.11, and 0.45±0.13 in well differentiated hepatic tissues. The immunohistochemical positive substance was detected in the plasma membrane and cytoplasm. The positive rates of P-gp, MRP1 and BCRP were 82.35%, 58.82%, and 79.41% in hepatocellular carcinoma and 42.11%, 26.32%, and 36.84% in paraneoplastic hepatic tissues, respectively. The positive rates of P-gp, MRP1 and BCRP were 100.00%, 81.25%, and 100.00% in poorly differentiated hepatocellular carcinoma and 66.67%, 38.89%, and 61.11% in well differentiated hepatic tissues. The expression of three indicies in hepatocellular carcinoma was higher than that in paraneoplastic hepatic tissues (P<0.05). The expression of P-gp/MDR1, MRP1 and BCRP in poorly differentiated hepatocellular carcinoma was higher than that in well differentiated hepatic tissues (P<0.05). No correlation was found among the three indices. Conclusion: Intrinsic multidrug resistance exsists in hepatocellular carcinoma, with various mechanisms. The multidrug resistance of HCC (hepatic cell carcinoma) is related to P-gp/MDR1, MRP1 and BCRP. MRP1 and BCRP may be targets for reversing multidrug resistance.

Objective To establish a new method for rapid detection of β-thalassemia by investigating six clinical common mutation types.Methods Fifty cases of clinical wild-type samples and 42 cases ofβ-thalassemia samples were collected, and β-globin gene was amplified by PCR.Uniform ligation probe ( ULP) specific probes were designed for hybridization reaction to increase the reaction specificity and real-time PCR was performed to increase the sensitivity.After that, PCR products were verified by agarose electrophoresis.After examining the specificity and sensitivity, Kappa test between LDR-ULP method and reverse dot blot( RDB) method was conducted.Results Hybridization efficiency was improved 2.53 times by LDR-ULP hybridization.Each mutant type showed a significant amplification curve, whereas the wild-type had no significant curve within 40 cycles.The limit of determination of this method was 5 pg.The results of 92 cases of peripheral blood samples detected by the method of LDR-ULP and RDB were completely consistent.Conclusion In this study, a simple, inexpensive, rapid new method to detect β-thalassemia were established.

OBJECTIVE: The dynamic response of the heart during chest impact and the characteristics of heart injuries were investigated to further understand the mechanisms of heart impact injuries. METHODS: Eleven dogs and thirty-four rabbits were subjected to front thoracic impact with different impact velocities and compression response. The accelerated movement of thoracic wall during the impact period was monitored. The pathological examination of the injured heart was done and the dynamic responses and mechanisms of injuries were analyzed with mathematics models. RESULTS: The analysis of mathematics model and experimental results showed that the injury severity of heart was well correlated with the viscous criterion. The thoracic wall was involved in bi-directional movement of compression and expansion. The injured heart showed spotty or stripy hemorrhages in the ventricle endocardium. Light microscopic examination showed interstitial bleeding and rupture of the myocardial fibers in the contusion area. The biomechanical analysis indicated that there was a large deformation caused by the stress concentration on the lateral ventricle wall. CONCLUSIONS: There is a high speed and excessive deformation of the heart during the impact period, which might be the key mechanism of heart injury. The strong impact and press coming from both sternum and vertebral column and the rapid elevation of pressure in the ventricle are the main cause of deformation.

Objective:To explore the genetic basis of a child with mental retardation and developmental delay.Methods:A child who had attended the genetic clinic of Linyi People′s Hospital from October 2023 to April 2024 was selected as the study subject. Intelligence and development were assessed with simplified Peabody scale. Electroencephalogram and imaging data were collected. Peripheral blood samples of the child and her parents were collected for the screening of genetic metabolic diseases, chromosomal karyotyping, and trio-whole genome sequencing (trio-WGS) analysis. Candidate variant was verified by Sanger sequencing, and RNAseq was carried out to verify the alternative splicing due to the candidate variant. This study has been approved by the Medical Ethics Committee of Linyi People′s Hospital (No. YX200083).Results:The patient was an 8-year-and-11-month-old girl. Both of her parents had normal phenotypes. The child was assessed by the simplified Peabody scale as having intellectual disability and developmental delay. MRI showed no definite abnormal signals within the brain parenchyma, and electroencephalogram was normal. Screening of genetic metabolic diseases showed no obvious abnormality. Chromosomal karyotype was normal. Trio-WGS has detected a c. 697+ 1G>A variant in the intron 4 of the NFIX gene, along with 9 other variants within eight genes. The c. 697+ 1G>A variant may cause abnormal splicing of the NFIX gene transcript. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c. 697+ 1G>A variant was predicted to be pathogenic (PVS1+ PS2+ PM2_Supporting), while the evidence for pathogenicity of the other 9 variants was insufficient. Conclusion:The novel de novo c. 697+ 1G>A variant of the NFIX gene probably underlay the pathogenesis of the child, which may have caused the disease by leading to abnormal splicing.

COVID-19 is caused by a novel coronavirus-severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), which has being spreading around the world, posing a serious threat to human health and lives. Neutralizing antibodies and small molecule inhibitors for virus replication cycle are the main antiviral treatment for novel coronavirus recommended in China. To further promote the rational use of antiviral therapy in clinical practice, the National Center for Infectious Diseases (Beijing Ditan Hospital Capital Medical University and the First Affiliated Hospital, Zhejiang University School of Medicine) invited experts in fields of infectious diseases, respiratory and intensive care to develop an Expert Consensus on Antiviral Therapy of COVID-19 based on the Diagnosis and Treatment Guideline for COVID-19 ( trial version 10) and experiences in the diagnosis and treatment of COVID-19 in China. The consensus is concise, practical and highly operable, hopefully it would improve the understanding of antiviral therapy for clinicians and provide suggestions for standardized medication in treatment of COVID-19.