BACKGROUND:The mechanism of differentiation and proliferation of bone marrow-derived mesenchymal stem cells remains unclear. In addition, issues such as how signal pathways such as Ca2+and bone marrow-derived mesenchymal stem cellproliferation and differentiation signals form complex signal network remain poorly understood.
OBJECTIVE:To investigate the effect of Ca2+in the induced differentiation of bone marrow-derived mesenchymal stem cells into hepatocytes.
METHODS:Bone marrow-derived mesenchymal stem cells were isolated from rat bone marrow using whone bone marrow adherence method, purified, amplified, and induced with hepatocyte growth factor. [Ca2+]i in the directional differentiated bone marrow-derived mesenchymal stem cells and control bone marrow-derived mesenchymal stem cells were detected with flow cytometry. Bone marrow-derived mesenchymal stem cells induced with hepatocyte growth factor were mixed with nimodipine of different concentration, and cells were divided into three groups:hepatocyte growth factor+nimodipine 10 mg/L, 50 or 100 mg/L groups. cellgrowth was observed with inverted phase contrast microscope and alpha 1-antitrypsin expression of the cells was confirmed by immunocytochemistry. The calcineurin M and the activation of extracellular signal regulated kinase pathway was detected by reverse transcription-PCR and western blotting, respectively.
RESULTS AND CONCLUSION:[Ca2+]i in the directional differentiated bone marrow-derived mesenchymal stem cells was higher than in the control group (P<0.05). After addition of a larger dose of nimodipine, no differentiation of cells was obeserved and growth of bone marrow-derived mesenchymal stem cells was getting worse. There were few alpha 1-antitrypsin positive cells in the nimodipine groups. Calcineurin Mexpression was significantly increased in directional differentiated bone marrow-derived mesenchymal stem cells and smal dose of nimodipine than the controls (P<0.05). However, no significant difference was found among middle, high dose nimodipine and control groups (P>0.05). These findings indicate that Ca2+could participate in the differentiation of bone marrow-derived mesenchymal stem cells into hepatocytes incuded with cytokines, and also maintain the survival and proliferation of bone marrow-derived mesenchymal stem cells.

Objective:To analyze the genetic test results and ultrasonographic markers of 41 fetuses with short femurs and their relationship.Methods:This study retrospectively analyzed 41 fetuses who were diagnosed with short femurs by ultrasound during 19-37 gestational weeks and underwent prenatal genetic examination at the First Affiliated Hospital of Zhengzhou University from January 2018 to June 2019. According to the results of genetic examination, these cases were divided into three groups after excluding three cases of variants of unknown significance: genetically normal group, chromosome variation (including chromosomal aneuploidy and pathogenic or likely pathogenic copy number variations) group, and gene mutation (including pathogenic or likely pathogenic gene mutations) group. According to the head circumference (HC), abdominal circumference (AC) and femur length (FL), Z FL, FL/HC, FL/AC, ΔZ H-F and ΔZ H+A-2F for each fetus were calculated. One-way ANOVA and LSD- t test were used for statistical analysis. Results:(1) Among the 41 fetuses with short femurs, there were 28 in the genetically normal group, five in the chromosome variation group, three with chromosome variations of unknown significance and five in the gene mutation group. (2) In the genetically normal, chromosome variation and gene mutation groups, Z FL values were -2.78±0.77, -4.36±0.69 and -4.69±0.70; FL/HC ratios were 0.178±0.011, 0.170±0.010 and 0.131±0.022; FL/AC ratios were 0.197±0.013, 0.186±0.011 and 0.151±0.017; ΔZ H-F values were 2.49±1.09, 3.53±1.28 and 8.17±1.30; ΔZ H+A-2F values were 4.44±2.00, 6.78±2.20 and 14.28±1.26, respectively. The differences in Z FL values between the genetically normal group and the chromosome variation group as well as the gene mutation group were statistically significant (both P<0.05); so were the differences in FL/HC, FL/AC and ΔZ H-F values between the gene mutation group and the genetically normal group as well as the chromosome variation group (all P<0.05) and in any pairwise comparison of ΔZ H+A-2F among the three groups (all P<0.05). Conclusions:The genetic etiology of fetal short femurs is mainly related to chromosomal variations (including chromosomal aneuploidy and pathogenic or likely pathogenic copy number variations) and gene mutation. In fetuses with chromosome variation and gene mutation, the degree of the femoral development delay relative to the development of HC and AC is worse than that in the normal genetic results group.

OBJECTIVE:To reduce patient waiting time for drugs by improving work efficiency via the optimization of the in-telligent workflow of outpatient pharmacy. METHODS:The problems existing in each link in the workflow after the adoption of au-tomated drug dispensing machine in the outpatient pharmacy of our hospital were analyzed. Measures were developed and imple-mented,on the basis of the factors affecting each link including adding drugs,making up a prescription and dispensing drugs,to optimize the workflow. The effect after the optimization was evaluated,with the time it takes to make up a prescription and to dis-pense drugs and patient waiting time for drugs as the indexes. RESULTS:The averaged time it takes a pharmacist to make up a pre-scription and to dispense drugs and patient waiting time for drugs significantly reduced from 3.4 min,9.3 min and 12.7 min to 1.0 min,6.1 min and 7.1 min respectively,after taking measures such as adjusting the number of the drug types,tracks and positions in the machine,adding the contrasting pictures of the drugs which were similar on the system interface for adding drugs,standardiz-ing the process of adding drugs,improving and allocating information labels,adjusting the process of dispensing drugs and adding new small packages of drugs within 6 months. CONCLUSIONS:The optimized intelligent workflow in the pharmacy can reduce pa-tient waiting time for drugs,increase patient satisfaction and promote the development of intelligent pharmacy.

OBJECTIVE To construct eukaryotic expression vectors for human ABO and subgroup genes A101, B101, CisAB01, Ael05, B(A)04 and Bw03, and validate their expression in vitro. METHODS Total RNA was isolated from individuals with the A101 and B101 subgroups. cDNA of A101 and B101 was synthesized by reverse transcription and amplified with specific primers. Subgroup genes CisAB01, Ael05, B(A)04 and Bw03 were then amplified with PCR for site-directed mutagenesis. Fragments of the ABO genes were directionally linked to pcDNA3.1 positive-eukaryotie expression vectors. After antibiotic screening, the sequences were analyzed. The vectors were transfected into Hela cells, and the expression of target proteins was detected by Western blotting and immunofluorescence assay. RESULTS Sanger sequencing has confirmed that pDNA3.1-A101, pDNA3.1-CisAB01, pDNA3.1-Ael05, pDNA3.1-B101, pDNA3.1- B(A)04, pDNA3.1-Bw03 positive-eukaryotic expression vector were successfully constructed. The results of Western blotting and immunofluorescence revealed clear presence of the expressed proteins. CONCLUSION Eukaryotic expression vectors for ABO subgroup genes were successfully constructed and worked well in Hela cells in vitro, which can facilitate further study of the ABO blood group proteins.

Objective To analyze and identify the pathogenic mutation that caused a case of child's renal coloboma syndrome (RCS).Methods A child with congenital cataract in the right eye and optic disc defect in the left eye and his parents with normal phenotype were included in the study.The blood of the child and his parents were captured to extract DNA and make molecular test.The possible variants were screened through NGS sequencing using the ophthalmology gene panel on illumina NextSeq 500 platform,and proved the selected PAX2 mutation by Sanger sequencing.Pathogenicity report was retrieved through PubMed and related database.Pathogenicity analysis of the candidate mutated site has careful consideration of the patient's clinical presentations and sequencing result base on Standards and Guidelines for the Interpretation of Sequence Variants revised by ACMG.According to the results of gene diagnosis,the child was executed related clinical examinations on kidney.Results The sequence result showed that a heterozygous mutation in PAX2,c.70dupG (p.V26Gfs*28),which lead to truncated protein product that terminated after 28 amino acids of the mutated site.Both of his normal parents were not carriers of the heterozygous mutation.Sanger sequencing results of the child and his parents were consistent with the NGS sequencing.The autosomal dominant disease phenotype was inferred to be caused by the heterozygous mutation of c.70dupG (p.V26Gfs*28) of PAX2 gene.Renal color Doppler ultrasound results showed the child with small renal cysts on the left and mildly separated collecting system.Renal function tests showed the child with αl microglobulin index increased.Conclusion The heterozygous mutation c.70dupG (p.V26Gfs*28) in PAX2 is the genetic pathogenic cause for the patient with RCS.

OBJECTIVE: To detect variants of EXT1 and EXT2 genes among five pedigrees affected with multiple osteochondromas and provide prenatal diagnosis for the families based on the results. METHODS: The EXT1 and EXT2 genes of the probands were analyzed by targeted next generation sequencing (NGS). Suspected pathological variants were validated by Sanger sequencing in the probands, their family members and 200 unrelated healthy controls. Multiple ligation-dependent probe amplification (MLPA) was used to confirm the presence of gross deletions. Prenatal diagnosis was provided for 2 couples carrying pathogenic or likely pathogenic variants. RESULTS: Five variants were detected in the pedigrees, which included EXT1 exon 2-3 deletion, c.1468dupC (p.Leu490ProfsX31), c.2084delC (p.Pro695LeufsX11), and EXT2 c.187delT (p.Phe63SerfsX29) and c.1362T>G (p.Tyr454X). Among these, EXT1 exon 2-3 deletion, c.2084delC (p.Pro695LeufsX11) and EXT2 c.187delT (p.Phe63SerfsX29) were unreported previously. The three novel variants were not found among unaffected members of the pedigree and the 200 healthy controls. Upon prenatal diagnosis, the two fetuses were found to carry the same variants of the the probands. CONCLUSION Pathological variants of the EXT1 and EXT2 genes probably underlie the multiple osteochondromas among the 5 pedigrees. Prenatal diagnosis based on the results can effectively reduce the birth of further offspring affected with the disease.

Objective:To analyze the variations of SGCA gene in two Chinese pedigrees of Han nationality with limb-girdle muscular dystrophy type 2D (LGMD2D) and provide prenatal diagnosis and genetic counseling for subsequent pregnancies within the pedigrees. Methods:This study involved two unrelated patients who were the probands of their pedigrees diagnosed with LGMD2D in the First Affiliated Hospital of Zhengzhou University from June 2017 to January 2018. Genomic DNA was extracted from peripheral blood samples of the probands and their parents. Coding sequences and flanking sequences of 21 LGMD-related genes from the probands were captured and subjected to high-throughput sequencing. Suspected mutations in their parents were detected and validated by Sanger sequencing and/or fluorescence quantitative polymerase chain reaction (PCR). Prenatal genetic diagnosis for high-risk fetuses in the two pedigrees was performed after the causative factors being identified.Results:(1) The proband of pedigree 1 carried compound heterozygous point mutations in SGCA gene with c.218C>G(p.P73R) and c.101G>A(p.R34H) inherited from his father and mother, respectively. Prenatal diagnosis indicated that the second fetus of the family carried the same mutations as the proband, and the family chose to terminate the gestation. (2) The proband of pedigree 2 inherited the compound heterozygous mutations of c.218C>T (p.P73L) and heterozygous deletion of exons 7 and 8 in SGCA gene from his parents. Their second fetus did not carry any of the above mutations and was delivered at full term. Serum creatinase level and physical, motor and mental development of the child were all within the normal range during a two-year follow-up after birth. Conclusions:The heterozygous mutations in SGCA gene are the cause of LGMD2D in the two pedigrees, and c.218C>G(p.P73R) and c.218C>T(p.P73L) are novel mutations. Genetic and prenatal diagnosis based on high-throughput targeted next-generation sequencing can rapidly and accurately detect the mutations responsible for LGMD2D.

ObjectiveTo investigate the risk factors for portal vein thrombosis (PVT) in cirrhotic patients with esophageal varices, and to establish a nomogram for predicting the risk of PVT. MethodsA retrospective analysis was performed for the clinical data of 283 cirrhotic patients with esophageal varices who attended Shandong Provincial Hospital Affiliated to Shandong University from December 2013 to December 2018, and according to imaging findings, the patients were divided into PVT group with 119 patients and non-PVT group with 164 patients. The t-test or the Mann-Whitney U test was used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between two groups. A multivariate logistic regression analysis was used to screen out independent risk factors; a nomogram was established and validated based on the results of the multivariate logistic regression analysis, and C-index and calibration curve were used to evaluate its performance. ResultsThe univariate analysis showed that compared with the non-PVT group, the PVT group had significantly higher Child-Pugh class (χ2=9.388, P=0.009), proportion of patients with a history of splenectomy (χ2=26.805, P＜0.001), white blood cell count (Z=-2.248, P=0.025), platelet count (Z=-3.323, P=0.001), D-dimer(Z=-6.236, P＜0.001), and spleen thickness (Z=-2.432, P=0.015) and a significantly lower level of triglyceride (TG) (Z=-4.150, P＜0.001). The multivariate logistic regression analysis showed that a reduction in TG (odds ratio ［OR］=0.441, 95% confidence interval ［CI］: 0.190-0.889), an increase in D-dimer (OR=1.151, 95%CI: 1.041-1.272), prolonged prothrombin time (PT) (OR=1160, 95%CI: 1.025-1.313), and a history of splenectomy (OR=2.933, 95%CI: 1.164-7.389) were independent risk factors for PVT in cirrhotic patients with esophageal varices. In addition, a nomogram was established based on the results of the multivariate regression analysis, with a C-index of 0.745, and the calibration curve showed good consistency between the observed and predicted values for the development of PVT. ConclusionA reduction in TG, an increase in D-dimer, prolonged PT, and a history of splenectomy are independent risk factors for PVT in cirrhotic patients with esophageal varices, and the nomogram developed based on these results can provide a quantitative and intuitive tool for clinicians to assess the risk of PVT.

OBJECTIVE: To identify the etiology of a patient with severe symptoms of DMD and to trace its pathogenic gene, so as to provide a basis for genetic counseling and clinical intervention. METHODS: Multiple ligation-dependent probe amplification (MLPA) technique was used to analyze exon deletion/repetitive variant of DMD gene, and further analysis was performed by chromosome G-banding, fluorescence in situ hybridization (FISH) and SNP array analysis. RESULTS: The MLPA results of the proband showed that the exon 1-79 of DMD gene were deleted, the G-banding karyotype of blood sample was 46, XY, and the deletion of the short arm of X chromosome was found by FISH. SNP array results showed that 5.8Mb (29 628 158-35 434 714) deletion occurred in the Xp21.2p21.1 region of X chromosome, and the patient was diagnosed as the contiguous deletion syndrome involving the genes of IL1RAPL, MAGEB1-4, ROB, CXorf2, GM, AP3K7IP, FTHL1, DMD, FAM47A, TMEM47, and FAM47B. CONCLUSION The exact pathogenic site of this family is the deletion of 5.8 Mb (29 628 158-35 434 714) in the Xp21.2p21.1 region of X chromosome, which can be used for prenatal diagnosis. High resolution SNP array technique plays an important role in detecting potential chromosome abnormalities in patients.
Dystrophin/genetics* ; Exons ; Female ; Gene Deletion ; Humans ; In Situ Hybridization, Fluorescence ; Muscular Dystrophy, Duchenne/genetics* ; Pregnancy ; Prenatal Diagnosis

OBJECTIVE: To carry out genetic testing and prenatal diagnosis for a Chinese couple whom had conceived two fetuses featuring multiple malformations including polycystic kidney, polydactyly and encephalocele. METHODS: Following elective abortion, the fetus from the second pregnancy was subjected to whole exome sequencing. Suspected pathogenic variants were verified by Sanger sequencing of the fetus and its parents. RESULTS: The fetus was found to harbor compound heterozygous variants of the CEP290 gene, namely c.2743G>T (p.E915X) and c.2587-2A>T, which were respectively inherited from its father and mother. The same variants were not detected among 100 healthy controls nor reported previously. Bioinformatic analysis suggested both variants to be deleterious. The fetus was diagnosed with Meckel-Gruber syndrome. Prenatal diagnosis for the couple during their next pregnancy suggested that the fetus did not carry the above pathogenic variants. CONCLUSION The compound heterozygous variants of the CEP290 gene probably underlay the pathogenesis of Meckel-Gruber syndrome in the second fetus. Above finding has provided a basis for genetic counseling and prenatal diagnosis for the couple, and also enriched the mutational spectrum of the CEP290 gene.
China ; Ciliary Motility Disorders ; Encephalocele/genetics* ; Female ; Genetic Testing ; Humans ; Pedigree ; Polycystic Kidney Diseases/genetics* ; Pregnancy ; Prenatal Diagnosis ; Retinitis Pigmentosa