Objective:To discuss the promotive effect of nerve growth factor(NGF),which is highly expressed in the adipose-derived stem cell(ADSC)-induced Schwann-like cells(SCLCs),on the growth of dorsal root ganglion(DRG)cell processes in the rats,and to clarify its mechanism.Methods:The ADSCs were extracted from the epididymal adipose tissue of the SD rats,and their multidirectional differentiation potential was identified through osteogenic,adipogenic,and chondrogenic induction.The ADSCs were induced to differentiate into the SCLCs,and the expression levels of glial fibrillary acidic protein(GFAP)and S100 calcium-binding protein β(S100β)protein in the ADSCs and SCLCs were detected by immunofluorescence staining and Western blotting methods.The DRG cells were isolated and cultured,and immunofluorescence staining was used to detect the βⅢ-tubulin expression in the DRG cells for identification.The SCLCs were co-cultured with the DRG cells(co-culture group),the single-culture DRG cells were regared as DRG group and toluidine blue staining was used to observe and measure the length of DRG cell processes under the optical microscope in co-culture group and DRG group.Small interfering RNA(siRNA)transfection was used to knock down NGF,and plasmid transfection was used to over-express NGF.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the NGF mRNA expression levels in the cells in various groups;enzyme-linked immunosorbent assay(ELISA)method was used to detect the NGF protein levels in the cell supernatants.The transfected SCLCs were co-cultured with DRG cells and divided into control group,siNC/vector group,NGF knockdown group(si-NGF group),and NGF over-expression group(oe-NGF group).The lengths of DRG cell processes in various groups were observed.Results:The primary ADSCs adhered within 24 h after seeding,with a small number of lipid droplets remaining.After 3 d of culture,the cells were mostly short spindle-shaped,fusiform,or polygonal,growing rapidly in a vortex pattern.After passaging,the cells exhibited a uniform morphology,appearing as long spindles arranged in a fish-school pattern.After 14 d of adipogenic induction,the cell morphology changed from spindle-shaped to flat-round,with translucent lipid droplets forming in the cytoplasm,which were stained red by Oil Red O.After 28 d of osteogenic induction,the cells appeared sand-like with blurred morphology,and calcified nodules were observed,which were stained red by Alizarin Red and deposited in the extracellular matrix.After 28 d of chondrogenic induction in a 3D culture system,millet-sized chondrogenic spheres formed.Frozen sections of the spheres were stained with Alcian Blue,and acidic mucopolysaccharides in the cartilage tissue were stained blue under the microscope.Under the fluorescence microscope,the third-passage purified ADSCs showed positive expression of CD29[fluorescein isothiocy anate(FITC)-labeled green fluorescence]and CD44(Cy3-labeled red fluorescence).The immunofluorescence staining results showed that GFAP was labeled with FITC(green fluorescence),and S100β was labeled with Cy3(red fluorescence).The Western blotting results showed that compared with ADSCs,the expression levels of S100β and GFAP proteins in the SCLCs were increased(P<0.05).The primary DRG cells began to adhere 6 h after conventional culture,and after 3 d,the cell bodies appeared round and bright,with two linear processes extending from them.Under fluorescence microscope,the cells positively expressed the neuron-specific marker βⅢ-tubulin,confirming that the isolated cells were DRG cells.Compared with the ADSCs,the NGF protein expression level in the SCLCs was increased(P<0.05).Compared with DRG group,the length of DRG cell processes in co-culture group was the highest when DRG cells and SCLCs were co-cultured at a 1∶2 ratio(P<0.05).The RT-qPCR results showed that compared with si-NC group,the expression levels of NGF mRNA in the cell supernatant in si-NGF-1,si-NGF-2,and si-NGF-3 groups were significantly decreased(P<0.05),with si-NGF-1 showing the highest knockdown efficiency,which was selected for subsequent experiments.The ELISA results showed that compared with si-NC group,the NGF levels in the cell supernatant of si-NGF-1,si-NGF-2,and si-NGF-3 groups were decreased(P<0.05).Compared with Vector group,the expression level of NGF mRNA and NGF protein level in the supernatant in oe-NGF group were increased(P<0.05).Compared with control group and siNC/vector group,the length of DRG cell processes in si-NGF group was decreased(P<0.05),while the length of DRG cell processes in oe-NGF group was increased(P<0.05).Conclusion:ADSCs can be directionally differentiated into SCLCs,and the differentiated cells highly express NGF.Knockdown or overexpression of NGF can affect the growth of DRG cell processes.

Objective:To observe the effects of adipose-derived stem cells(ADSC)combined with acellular scaffold(AS)on the ultrastructure of dorsal root ganglion and the protein and mRNA expression levels of ciliary neurotrophic factor(CNTF),Janus kinase 2(JAK2),phosphorylated JAK2(p-JAK2),signal transducer and activator of transcription 3(STAT3)and phosphorylated STAT3(p-STAT3)in the rats with sciatic nerve injury(SNI),and to clarify the protective effect of ADSC combined with AS on dorsal root ganglion in the SNI rats and its possible mechanism.Methods:The rat ADSCs were isolated and cultured and their multidirectional differentiation potential was detected.The AS of rats was prepared,and ADSCs were injected into the AS to construct tissue-engineered nerve.A total of 36 rats were randomly divided into control group,model group,AS group,and ADSC+AS group.The rats in control group were routinely fed,and the rats in other groups were used to establish the SNI models by resecting 10 mm of right sciatic nerve.The rats in model group received no further treatment,while the rats in AS group and ADSC+AS group were bridged with AS and the constructed tissue-engineered nerve at the two ends of the injured nerve,respectively.At 6 weeks after surgery,transmission electron microscope was used to observe the ultrastructure of dorsal root ganglion of the rats in various groups;immunofluorescence method was used to detect the protein expression levels of CNTF,p-JAK2,and p-STAT3 in dorsal root ganglion of the rats;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the mRNA expression levels of CNTF,JAK2,and STAT3 in dorsal root ganglion of the rats in various groups.Results:After 7 d of primary ADSC culture,a large number of large and long spindle-shaped cells were observed under the inverted microscope,arranged in clusters or whirlpools;red lipid droplets were observed with oil red O staining under microscope,and calcified nodules were observed with Alizarin red staining under microscope,indicating that the isolated and cultured cells had multidirectional differentiation ability.Compared with normal nerve tissue,the level of DNA in AS of rats was significantly decreased(P<0.05).Compared with control group,the nuclear membrane of dorsal root ganglion cells in model group was uneven and serrated,the number of organelles in the cytoplasm was decreased,mitochondria were swollen with broken or missing cristae and unclear structure;the CNTF protein and mRNA expression levels were significantly decreased(P<0.01),the p-JAK2 and p-STAT3 protein expression levels were significantly increased(P<0.01),and the JAK2 and STAT3 mRNA expression levels were significantly increased(P<0.01).Compared with model group,the serrated change of nuclear membrane of the dorsal root ganglion cells in AS group was significantly alleviated,the number of organelles in the cytoplasm was increased,and mitochondrial swelling was reduced;in ADSC+AS group,the nuclear membrane of dorsal root ganglion cells tended to be intact,the number of organelles was increased,and mitochondrial swelling and vacuolization were significantly reduced;the CNTF protein and mRNA expression levels in the dorsal root ganglion in AS group and ADSC+AS group were significantly increased(P<0.01),the p-JAK2 and p-STAT3 protein expression levels were significantly decreased(P<0.01),and the JAK2 and STAT3 mRNA expression levels were significantly decreased(P<0.01).Compared with AS group,the CNTF protein and mRNA expression levels in ADSC+AS group were significantly increased(P<0.05 or P<0.01),the p-JAK2 and p-STAT3 protein expression levels were significantly decreased(P<0.01),and the JAK2 and STAT3 mRNA expression levels were significantly decreased(P<0.01).Conclusion:The application of ADSC combined with AS can improve the ultrastructure of dorsal root ganglion in the SNI rats,and the mechanism may be related to the increased CNTF expression and decreased activation of the JAK2/STAT3 signaling pathway in the dorsal root ganglion by ADSC combined with AS application.

Objective:To summarize the ultrasonographic characteristics, genetic etiology, and perinatal prognosis of fetuses with absence of ductus venosus (ADV).Methods:A retrospective study enrolled 84 singleton pregnancies that underwent prenatal ultrasound examination and were diagnosed with fetal ADV at the First Affiliated Hospital of Zhengzhou University from June 2017 to July 2022. Based on prenatal ultrasonographic findings, the cases were divided into isolated ADV group ( n=37), ADV with ultrasound soft markers group ( n=9), and ADV with definite ultrasound abnormalities group ( n=38). According to the gestational age at the initial diagnosis of ADV, they were categorized into early pregnancy group (11-13 weeks of 6 days) with 17 cases, mid-pregnancy group (14-27 weeks of 6 days) with 45 cases, and late pregnancy group (≥28 weeks) with 22 cases. Depending on the direction of blood flow in the intra-abdominal segment of the umbilical vein, they were classified into umbilical vein directly entering the portal sinus group ( n=75), intrahepatic umbilical vein abnormal shunt group ( n=4), and extrahepatic umbilical vein shunt group ( n=5). The clinical characteristics of each group were summarized, and compared using the Chi-square, trend Chi-square tests, Fisher's exact test and Bonferroni correction test. Results:The common ultrasonographic abnormalities in the 84 cases of ADV fetuses were cardiac anomalies (27.4%, 23/84), cystic hygroma (10.7%, 9/84), fetal hydrops (9.5%, 8/84), and body cavity effusion (8.3%, 7/84). The proportions of fetuses with ADV and definite ultrasound abnormalities detected in the early, mid, and late pregnancy were 16/17, 44.4% (20/45), and 9.1% (2/22), respectively, with a higher proportion of definite ultrasound abnormalities associated with earlier detection of ADV ( χ 2trend=27.25, P<0.001). Among them, 21 cases underwent chromosomal karyotyping and/or chromosomal copy number variation sequencing or expanded non-invasive prenatal testing, with five abnormalities detected, including 45,X, trisomy 13, trisomy 22 mosaicism, trisomy 7 mosaicism, and a 14 Mb duplication at 22q12.3q13.33. The neonatal survival (28 days after birth) rates with ADV detected in the early, mid, and late pregnancy gradually increased, at 1/17, 43.9% (18/41), and 90.5% (19/21), respectively ( χ 2trend=27.04, P<0.001). The neonatal survival rates of the isolated ADV group and the group with ultrasound soft markers were higher than that of the group with definite ultrasound abnormalities [93.9% (31/33) and 6/9 vs. 2.7% (1/37), Bonferroni corrected, both P<0.001]. The neonatal survival rates of the umbilical vein directly entering the portal sinus group, intrahepatic umbilical vein abnormal shunt group, and extrahepatic umbilical vein shunt group were 50.0% (35/70), 0/4, and 1/5, respectively, with no statistically significant difference (Fisher's exact test, P=0.105). Conclusions:The earlier the detection of fetal ADV, the more likely it is to be associated with definite ultrasound abnormalities and have lower neonatal survival rates. This highlights the importance of ultrasonographic examination of the fetal ductus venosus. Once ADV is detected, attention should be paid to other potential ultrasound abnormalities, and genetic testing should be completed.

OBJECTIVE: Flavonoids are the bioactive compounds in safflower (Carthamus tinctorius), in which chalcone synthase (CHS) is the first limiting enzyme. However, it is unclear that which chalcone synthase genes (CHSs) are participated in flavonoids biosynthesis in C. tinctorius. In this study, the CHSs in the molecular characterization and enzyme activities were investigated. METHODS: Putative chalcone biosynthase genes were screened by the full-length transcriptome sequences data in C. tinctorius. Chalcone biosynthase genes in C. tinctorius (CtCHSs) were cloned from cDNA of flowers of C. tinctorius. The cloned gene sequences were analyzed by bioinformatics, and their expression patterns were analyzed by real-time PCR (RT-PCR). The protein of CtCHS in the development of flowers was detected by polyclonal antibody Western blot. A recombinant vector of CtCHS was constructed. The CtCHS recombinant protein was induced and purified to detect the enzyme reaction (catalyzing the reaction of p-coumaryl-CoA and malonyl-CoA to produce naringin chalcone). The reaction product was detected by HPLC and LC-MS. RESULTS: Two full-length CtCHS genes were successfully cloned from the flowers of safflower (CtCHS1 and CtCHS3), with gene lengths of 1525 bp and 1358 bp, respectively. RT-PCR analysis showed that both genes were highly expressed in the flowers, but the expression of CtCHS1 was higher than that of CtCHS3 at each developmental stage of the flowers. WB analysis showed that only CtCHS1 protein could be detected at each developmental stage of the flowers. HPLC and LC-MS analyses showed that CtCHS1 could catalyze the conversion of p-coumaryl-CoA and malonyl-CoA substrates to naringin chalcone. CONCLUSION CtCHS1 is involved in the biosynthesis of naringin chalcone in safflower.

OBJECTIVE: To carry out prenatal diagnosis for a fetus with normal ultrasonographic finding at 20 weeks' gestation but a copy number variant(CNV) of 13q indicated by non-invasive prenatal test (NIPT). METHODS: Karyotyping analysis and chromosomal CNV assay were carried out on the amniotic fluid sample. Parental peripheral blood sample was collected for chromosomal analysis. Detailed fetal ultrasound scan was carried out to rule out structural abnormalities of the fetus. RESULTS: The fetus was detected with a heterozygous 10.14 Mb deletion at 13q21.1q21.32, which has originated from the phenotypically normal mother. No apparent karyotypic abnormality was detected in the fetus and its parents. No ultrasonic abnormality was found in the fetus. CONCLUSION Both the fetus and its mother have carried a heterozygous 10.14 Mb deletion at 13q21.1q21.32 and presented normal phenotypes.Combined with literature review, the segmental deletion was judged to be a benign variant.
Female ; Genetic Counseling ; Humans ; Karyotyping ; Pedigree ; Pregnancy ; Prenatal Diagnosis ; Ultrasonography, Prenatal

OBJECTIVE: To explore the genetic etiology in four patients with hyperbilirubinemia, and discuss the correlation between clinical characteristics and molecular basis. METHODS: The data of clinical manifestation and auxiliary examinations were collected. Genomic DNA of the four patients was extracted and analyzed by next-generation sequencing using the panel including genes involved in hereditary metabolic liver diseases. Suspected variants were verified by Sanger sequencing. RESULTS: All of the four patients were males with normal liver enzymes. It was revealed that all the patients had heterozygous variants, among which c.3011C>T, c.2443C>T and c.2556del were the variants which have not been reported previously. CONCLUSION All of the patients were diagnosed as Dubin-Johnson syndrome (DJS) caused by ABCC2 gene variants. The novel variants add to the spectrum of genetic variants of the disease. Because of the favorite prognosis, precise diagnosis can greatly reduce the psychological pressure of patients and avoid excessive treatments. At the same time, it could provide pertinent genetic counseling for the families.
DNA ; Female ; Heterozygote ; Humans ; Jaundice, Chronic Idiopathic/genetics* ; Male ; Multidrug Resistance-Associated Protein 2 ; Multidrug Resistance-Associated Proteins/genetics* ; Phenotype

Objective:To analyze the clinical features and genetic basis for a child featuring elevated creatine kinase (CK).Methods:Next-generation sequencing (muscular dystrophy-related gene panel) was carried out for the proband. Candidate variants were verified by Sanger sequencing of the child and his parents.Results:The child was found to harbor compound heterozygous variants of the FKTN gene, including a missense c. 536G>C (p.R179T) variant from his father and a non-frameshift c. 1299_1301delGTG (p.W434del) variant from his mother. Both variants were predicted to be pathogenic. Conclusion:The compound heterozygous variants of the FKTN gene probably underlay the disease in this child. Above finding has expanded the mutation spectrum of congenital muscular dystrophy.

Objective:To explore the value of radiomics based on 18F-fluorodeoxyglucose (FDG) PET/CT in predicting the Children′s Oncology Group (COG) risk stratification of neuroblastoma (NB). Methods:From March 2018 to November 2019, the 18F-FDG PET/CT images of 125 NB children (51 males, 74 females, age: 0.5-10.5 years) confirmed pathologically in Beijing Friendship Hospital were retrospectively analyzed. According to the COG classification, patients were divided into high-risk group and non-high-risk group (including low- and intermediate-risk). Imaging radiomics features were extracted from PET and CT images and screened. Logistic regression was used to build the first model based on radiomics features (R_model) and calculate radiomics score (Rad_score), then build the second model (RD_model) based on Rad_score and demographic features and at last build the third model (RDC_modle) based on Rad_score, demographic features and clinical features. The receiver operating characteristic (ROC) curve was used to evaluate the predictive efficacy of these models. Results:The training set contained 94 NB cases (63 high-risk cases, 31 non-high-risk cases), and the validation set contained 31 NB cases (21 high-risk cases, 10 non-high-risk cases). Four radiomics features were obtained by screening, of which two features were based on CT images and the other two features were based on PET images. The area under the curves (AUCs) of the R_model, RD_model and RDC_model in training or validation set were 0.91, 0.94, 0.98 or 0.86, 0.92, 0.95, respectively. The accuracies of the R_model, RD_model and RDC_model in training or validation set were 86%(81/94), 89%(84/94), 93%(87/94) or 84%(26/31), 84%(26/31), 87%(27/31), respectively.Conclusions:Radiomics based on 18F-FDG PET/CT can accurately predict the COG risk stratification of NB. Prediction model of radiomics features combined with demographic and clinical characteristics can further improve the accuracy of predicting NB COG risk stratification, which can help personalized and precise therapy protocol management in NB.

Objective:To disclose the expression and correlation of E6, E7 and PD-L1 in cervical squamous cell carcinoma (CSCC) tissues and explore the immune regulation of E6, E7 on PD-L1.Methods:The expressions of E6, E7 and PD-L1 in human papillomavirus (HPV) negative normal cervical tissue, cervical intraepithelial neoplasia (CIN) tissue and HPV16, 52 and 58 positive CSCC tissue were detected by Western blot (WB). HPV16 positive CSCC and HPV negative CSCC tissues were used for primary cell isolation, identification and culture. E6, E7 small interfering RNAs (siRNAs) were constructed and transfected into HPV16 positive CSCC cells via liposomes.The expression of PD-L1 in the cells was detected by WB. Plasmids of E6, E7 gene were constructed and overexpressed in HPV negative CSCC cells to detect the expression of PD-L1.Results:The expression of E6, E7 and PD-L1 was not detected in the HPV negative normal cervical. While in the HPV16, 52, 58 positive CSCC tissue group, the expression of all the three were significantly higher than that in the CIN group ( P<0.01). Primary HPV16 positive CSCC and HPV negative CSCC cells were successfully isolated and cultured, which were named as H16CC cell lines and HNCC cell lines respectively. After inhibiting the expression of E6, E7 in H16CC cell lines, the expression of PD-L1 was significantly decreased ( P<0.05). The expression of PD-L1 was significantly increased after the overexpression of E6, E7 in HNCC cell lines ( P<0.05). Conclusions:The expression of PD-L1 is positively correlated with E6, E7, which may regulate the occurrence and development of CSCC by mediating the immune escape mechanism of PD-L1 in CSCC.

Objective:To explore the effect of plastic surgery suture technique and its proficiency in facial scar inhibition after trauma, and to explore the key factors to improve the suture proficiency of junior residents.Methods:The data of patients with facial trauma who underwent plastic surgery suture in the Department of Plastic and Reconstructive Surgery of Ningbo First Hospital from June 2017 to July 2019 were retrospectively analyzed. They were divided into senior group and junior group according to the seniority of chief surgeon. The general condition, scar appearance and local symptoms of the two groups were evaluated by the scar cosmesis assessment and rating scale(SCAR), including scar expansion, erythema, hyperpigmentation or hypopigmentation, suture marks, hyperplasia or atrophy, scar pruritus, scar pain, and the results were statistically analyzed.The mean of continuous data were calculated and expressed as Mean ± SD, the differences between groups were tested by t-test, and the classified data were expressed by rate, and the differences between groups were tested by chi-square test. Results:A total of 83 patients (54 females and 29 males) were included in this study, the maximum age was 63, the minimum age was 3, and the average age was 31.7±13.3 years old, including senior group (52 cases) and junior group (31 cases). The differences were not statistically significant in gender, age, injury time, wound length and complications between the two groups. The total scores of SCAR scale in the senior and junior groups were 2.18±0.98 and 2.78±1.30, respectively, the difference was statistically significant ( P=0.020). The senior group was better than the junior group in inhibiting scar expansion ( P=0.035), eliminating suture marks ( P=0.018), overall scar impression ( P=0.038) and reducing pigment abnormality ( P=0.045). However, in inhibiting erythema and inhibiting scar hyperplasia or atrophy, the differences were not statistically significant between two groups. In the senior group, 4 patients had pain within 24 hours, 3 patients had pruritus; in the junior group, 2 patients had pain, 3 patients had pruritus. Conclusions:Plastic surgery suture technique will effectively improve the appearance of facial scar after trauma, especially in inhibiting scar expansion, erythema, hyperplasia or atrophy, and overall impression.Junior doctors can be competent for this work to a certain extent, but thay need long-term training to master the technology, and skilled operation can further improve the curative effect.