Effects of 5-Aza-dC combined with chemotherapy regimens on the apoptosis of lung adenocarcinoma cells / 中国综合临床

Objective:To investigate the effect of different chemotherapy drugs combined with DNA methylase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-dC) on the apoptosis of lung adenocarcinoma cells.Methods:In the prospective randomized controlled study, lung adenocarcinoma A549 cells were treated with cisplatin plus paclitaxel (TP) or gemcitabine (GP) with or without 5-Aza-dC. According to different drug intervention methods, they were divided into control group, cisplatin combined with paclitaxel (TP) group, cisplatin combined with gemcitabine (GP) group, and 5-Aza-dC combined with TP group, 5-Aza-dC combined with GP group. CCK-8 assay was used to detect the proliferation of A549 cells. Transwell migration and invasion assay were used to detect the effect that each group of drugs on the migration and invasion ability of A549 cells. Quantitative Real-time Polymerase Chain Reaction was used to evaluate the effect of each treatment on the expression of apoptotic genes. One-way analysis of variance was used to compare the degree of cell proliferation in different drug treatment groups, and LSD- t method was used for pairwise comparison within groups. Results:The inhibition rates of lung adenocarcinoma cells in the TP regimen at different time points at 24, 48, and 72 h were as follows (20.00±4.23) %, (35.00±2.80) %, and (56.00±3.11) %. The inhibition rate of 5-Aza-dC combined with TP regimen on lung adenocarcinoma cells was significantly increased, at different time points of 24, 48 and 72 h, respectively (38.00±3.80) %, (50.00±3.25) %, (93.00±4.33) %. The inhibition rates of cells at different time points at 24, 48, and 72 h in the GP regimen were (33.00±5.10) %, (54.00±3.80) %, and (74.00±2.82) %, respectively; while 5-Aza-dC combined with GP regimen could significantly reduce the rate of cell growth, the inhibition rates of cells at 24, 48, and 72 h different time points were as follows (54.00±3.00) %, (67.00±5.30) %, and (95.00±1.13) %. The inhibitory effect of the same drug on lung adenocarcinoma cells increased with time (TP group: F=35.93, P<0.001; 5-Aza-dC combined with TP group: F=97.33, P<0.001; GP group: F =41.73, P<0.001; 5-Aza-dC combined with GP group: F=79.00, P<0.001), and at different time points, the differences were statistically different (all P<0.05). 5-Aza-dC combined with TP and GP chemotherapy regimens can inhibit the migration and invasion of lung adenocarcinoma cell A549, and the inhibitory effect is stronger than that of TP or GP regimens alone. The expression of Caspase 8 was significantly elevated ( t=5.87, P=0.004) in cells treated with 5-Aza-dC combined with GP when compared with GP regimen alone. The expression of Caspase 8 ( t=3.94, P=0.017), Caspase 6 ( t=5.81, P=0.004) and BBC3 (BCL-2 binding component 3) ( t=6.53, P=0.003) were increased when drugged with 5-Aza-dC combined TP regimen compared with TP regimen alone. Conclusion:5-Aza-dC might serve as a chemotherapeutic sensitizer to increase the sensitivity of lung adenocarcinoma cells.

Clinical features and gene mutation analysis of a male case of subcortical band heterotopia caused by mosaic mutation of DCX gene / 中华神经科杂志

Objective:To investigate the clinical phenotype and genotype of a male case of subcortical band heterotopia caused by mosaic mutation of DCX gene.Methods:The clinical data and magnetic resonance imaging (MRI) features of a male case of subcortical band heterotopia diagnosed in the Department of Neurology, Children′s Hospital Affiliated to Zhengzhou University in August 2020 were analyzed retrospectively. At the same time, the whole exon sequencing of the families was performed by next generation sequencing method, the suspicious mutation was verified by polymerase chain reaction Sanger sequencing, and their genetic mutation characteristics were analyzed.Results:The proband, one male, aged 5 years and 1 month, was hospitalized in August 2020 with the complaint of intermittent convulsions for 4 years and six months. Clinical features included that limb muscle tension was slightly high, intellectual and motor development was backward, and head circumference was 48 cm. MRI of his head showed diffuse thick subcortical band heterotopia. The detection of whole exon sequencing in his family showed that there was hemizygous mosaic mutation in DCX gene (mosaic ratio 44%), c.148A>G (p.k50E). The mosaic ratios of oral mucosa and urinalysis were 38.2% and 44.8% respectively. His parents were wild-type, The mutation found in this patient has not been reported at home and abroad.Conclusions:The mosaic variation of DCX gene can cause subcortical band heterotopia in males. The variation of DCX gene c.148A>G (p.k50E) may be the possible cause of the proband, which expands the variation spectrum of subcortical band heterotopia.

Dihydromyricetin mediates epithelial mesenchymal transformation and regulates the proliferation and apoptosis of esophageal squamous cell carcinoma cells / 中华肿瘤杂志

Objective: To study the effects of dihydromyricetin (DMY) on the proliferation, apoptosis and epithelial mesenchymal transition (EMT) of esophageal squamous cell carcinoma (ESCC) cell KYSE150 and KYSE410. Methods: KYSE150 and KYSE410 cells were treated with different concentrations of DMY (0, 25, 50, 100, 150, 200 μmol/L) for 24 hours. The median inhibition concentration (IC50) values of KYSE150 and KYSE410 were detected by cell counting kit-8 (CCK-8) method. Then 0.5‰ dimethyl sulfoxide (DMSO) was used as control group, dihydromyricetin (DMY), dihydromyricetin and transforming growth factor-β1 (DMY+ TGF-β1), transforming growth factor-β1 (TGF-β1) were used as experimental group. Cell proliferation and apoptosis rates were measured by clonal formation and flow cytometry. Transwell invasion and wound healing assay were used to detect cell invasion and migration. The protein expression levels of Caspase-3, Caspase-9, Bcl-2, Bax, Smad2/3, phosphorylation-Smad2/3 (p-Smad2/3) and Vimentin were detected by western blot. Results: The IC50 values of DMY on KYSE410 and KYSE150 cells were 100.51 and 101.27 μmol/L. The clone formation numbers of KYSE150 and KYSE410 in DMY group [(0.53±0.03) and (0.31±0.03)] were lower than those in DMSO group [(1.00±0.10) and (1.00±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in DMY group [(1.84±0.22)% and (2.80±0.07)%] were higher than those in DMSO group [(1.00±0.18)% and (1.00±0.07)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in DMY group [(0.42±0.03) and (0.29±0.05)] were lower than those in DMSO group [(1.00±0.08) and (1.00±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in DMY group [(0.65±0.14)% and (0.40±0.17)%] were lower than those in DMSO group [(1.00±0.10)% and (1.00±0.08)%, P<0.05]. The clone formation numbers of KYSE150 and KYSE410 in TGF-β1 group [(1.01±0.08) and (0.99±0.25)] were higher than those in DMY+ TGF-β1 group [(0.73±0.10) and (0.58±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in TGF-β1 group [(0.81±0.14)% and (1.18±0.10)%] were lower than those in DMY+ TGF-β1 group [(1.38±0.22)% and (1.85±0.04)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in TGF-β1 group [(1.19±0.11) and (1.39±0.11)] were higher than those in DMY+ TGF-β1 group [(0.93±0.09) and (0.93±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in TGF-β1 group [(1.87±0.19)% and (1.32±0.04)%] were higher than those in DMY+ TGF-β1 group [(0.86±0.16)% and (0.77±0.12)%, P<0.05]. The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY group were higher than those in DMSO group, while the protein expression level of Bcl-2 was lower than that in DMSO group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in DMY group were lower than those in DMSO group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in TGF-β1 group were lower than those in DMY+ TGF-β1 group, and the protein expression level of Bcl-2 was higher than that in DMY+ TGF-β1 group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY+ TGF-β1 group were lower than those in DMY group, and the protein expression level of Bcl-2 was higher than that in DMY group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in TGF-β1 group were higher than those in DMY+ TGF-β1 group (P<0.05). Conclusion: DMY can inhibit the proliferation and EMT of ESCC mediated by TGF-β1 and promote cell apoptosis.

Research progress in antisense drug mechanism and chemical modification strategies / 药学学报

Over the past three decades, more and more antisense drugs have been approved for marketing or clinical trails. Antisense technology has become the focus of pharmaceutical research due to its unique advantages in treating diseases and strong clinical development potential. There is a big difference from traditional small molecule chemical drugs, and macromolecular protein biological drugs. Antisense drugs are a very independent drug form. Antisense drugs were initially used to treat diseases with single gene mutations, but recently they have gradually begun to be used for the treatment of common diseases. Rational antisense drug design is crucial for disease treatment based on genetics. This paper reviews the latest progress in the field of action mechanism, chemical modification and delivery strategy of antisense drugs, and analyzes the current intractable problems. It is believed that with the resolution of these problems, the research of antisense drugs can reach a new level.

Clinical and genetic analysis of two rare male patients with Rett syndrome / 中华医学遗传学杂志

OBJECTIVE@#To conduct clinical and genetic analysis of two male patients with atypical Rett syndrome.@*METHODS@#Collection of clinical data in the two patients and these parents; whole exome sequencing (WES) was used to detect the potential variants, which were verified by Sanger sequencing. X chromosome inactivation (XCI) detection is performed in the Patient 1's mother to detect the allelic expression difference of the MECP2 gene.@*RESULTS@#Patient 1, a 5-year and 10-month-old boy, had mental disorders and mild intellectual disability (ID) (IQ: 54), whose mother had ID. Patient 2 was a 9-month and 18-day-old male presented with recurrent infections, respiratory insufficiency, hypotonia and global developmental delay. WES indentified a hemizygous mutation, c.499C>T (p.R167W), in the MECP2 gene in patient 1, which was inherited from his mother. The inactivation of X chromosome is skewed, and the expression ratio of wild-type and mutant MECP2 is 100%:0. Patient 2 was found a de novo splicing mutation, c.62+2_62+3del in the MECP2 gene. They were both reported pathogenic variant related to Rett syndrome. c.499C>T (p.R167W) was defined as likely pathogenic (PS1+PM2+PP3) and c.62+2_62+3del was pathogenic (PVS1+PM2+PM6) based on American College of Medical Genetics and Genomics standards and guidelines.@*CONCLUSION@#Both the two patients were diagnosed with rare male Rett syndrome, which had atypical clinical manifestations and large difference. Above foundings have revealed novel phenotypes in Chinese male patients with Rett syndrome.

Clinical characteristics and genetic analysis of a Chinese pedigree affected with mitochondrial DNA depletion syndrome due to compound heterozygous variants of RRM2B gene / 中华医学遗传学杂志

OBJECTIVE@#To analyze the clinical characteristics and pathogenic gene in a Chinese pedigree affected with mitochondrial DNA depletion syndrome 8A (MTDPS8A).@*METHODS@#Whole exome sequencing was carried out for the patient. Sanger sequencing was used to verify the results, and PolyPhen-2 and PROVEAN software were used to predict the impact of amino acid changes on the function of the protein.@*RESULTS@#The patient, a two-month-old female, was admitted to the hospital for poor milk intake and poor mental response. Her clinical manifestations included feeding difficulty, shortness of breath and low muscle tone. Auxiliary laboratory test indicated that the infant was underdeveloped with abnormal liver, kidney, and heart functions accompanied by hyperlacticacidemia. She responded poorly to treatment and eventually died. Sequencing revealed that the child has carried compound heterozygous missense variants of the RRM2B gene, namely c.16delA (p.R6Gfs*22) and c.175G>C (p.A59P), which were respectively inherited from her father and mother, and both were newly discovered pathologic variants.@*CONCLUSION@#The c.16delA and c.175G>C compound heterozygous variants of the RRM2B gene probably underlay the pathogenesis of MTDPS8A. Above finding has strengthened the understanding of the clinical feature and genetic etiology of this disease and expanded the mutation spectrum of the RRM2B gene.

Case report of pulmonary hypertension caused by filamin A mutation / 中华实用儿科临床杂志

The clinical data of a case of pulmonary arterial hypertension (PAH) caused by mutation of filamin A( FLNA) in the congenital heart disease center of Wuhan Asian Heart Hospital in September 2017 were subject to retrospective analysis.A 2-year-old girl had clinical characteristics of special face, backward growth and development, repeated pulmonary infections, repeated heart failure, congenital heart disease, and PAH.Genetic detection: FLNA mutation (exon34: c.5417-1G> A), which was a new pathogenic mutation.There were few reports of PAH caused by FLNA mutations.In this study, a new case of pulmonary hypertension caused by FLNA c. 5417-1G>A compound heterozygous shearing mutations was found, which enriched the FLNA mutation spectrum and prompted genetic detection was an important approach to detect the cause of PAH.

Protective effect of Portulaca oleracea L. extract on acute liver injury induced by carbon tetrachloride in mice and its chemical composition analysis / 药学实践杂志

Objective To investigate the protective effect of the ethanol extract of Portulaca oleracea L. on acute liver injury induced by carbon tetrachloride in mice, and to analyze its effective components. Methods 80% ethanol purslane extract was centrifuged, vacuum distillated and vacuum dried into whole plant extract, supernatant extract and precipitated extract. Eighty ICR male mice were randomly divided into 8 groups: control group, liver injury model group, whole plant extract low-dose group, high-dose group, supernatant extract low-dose group, high-dose group, precipitation extract low-dose group, and high-dose group. After oral administration of distilled water or three kinds of purslane extract suspensions at different doses for 1 week, olive oil or CCl4 olive oil solution were injected subcutaneously respectively. After 16 hours, serum was collected to detect the levels of ALT, AST and IL-6 to evaluate the protective effect of purslane on acute liver injury. Ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS) was used to analyze the effective components of purslane extract. Results Compared with the model group, the levels of serum AST, ALT and IL-6 in high-dose whole plant extract group were significantly reduced. The serum ALT level of mice in the high-dose precipitation extract group was significantly reduced (P<0.05). The serum IL-6 level was decreased, but there was no significant difference. There were no significant changes in the levels of serum AST, ALT and IL-6 in the other intervention groups. 15 main components such as malic acid, citric acid, leucine, isoleucine, adenosine, succinic acid, genistein, tyrosine and phenylalanine were identified by UPLC-Q-TOF/MS. Conclusion Purslane whole plant ethanol extract has hepatoprotective and anti-inflammatory effects on CCl4 acute liver injury mice, which may be a combined effect of 15 active components.

Clinical phenotype and genetic analysis of pyridoxine dependent epilepsy induced by aldehyde dehydrogenase 7 family member A1 gene mutation / 中华神经科杂志

Objective:To investigate the clinical phenotypes, therapy and genetic features of aldehyde dehydrogenase 7 family member A1 (ALDH7A1) gene mutations in five cases of pyridoxine dependent epilepsy (PDE) with diagnosis confirmed by next generation sequencing.Methods:Retrospective analysis was carried out on clinical data of five cases of PDE children with early epilepsy onset who were treated in the Department of Neurology of Children′s Hospital Affiliated to Zhengzhou University from February 2018 to November 2019. Next generation sequencing approach was used for genetic sequencing of proband ALDH7A1 gene and the first generation Sanger was used for validation of family members. And the characteristics of gene mutations were analyzed.Results:Among the five children diagnosed with PDE, the male to female ratio was 4 ∶ 1 and ages at clinic visit ranged from two months to 10 months old. In clinical phenotypes, all five cases experienced onset in neonatal period, with repeated seizures, manifested as myoclonus, spasms or focal paroxysm. The administration of antiepileptic drugs performed poorly in seizure control while long term oral intake of large dose pyridoxine showed better efficacy. All the five cases of children came from compound heterozygous mutations of father and mother, i.e. slicing homozygous mutation c.247-2(IVS2)A>T, missense mutation c.584A>G (p.N195S) and nonsense mutation c.1003C>T(p.R335 *), missense mutation c.1553G>C(p.R518T) and c.1547A>G(p.Y516C), missense mutation c.1547A>G(p.Y516C) and frameshift mutation c.1566_1568delTAC, missense mutation c.1061A>G(p.Y354C) and nonsense mutation c.841C>T(p.Q281X, 259), among which c.247-2(IVS2)A>T was novel splicing site mutation not reported before. Conclusions:PDE is induced by ALDH7A gene mutation. Early clinical manifestations are mostly onset of refractory epilepsy in neonatal period. Antiepileptic drugs perform poorly in terms of efficacy while pyridoxine can control seizure effectively. Gene analysis should be conducted on such patients for confirmed diagnosis.

MRI features and signal pattern of primary sinonasal malignant melanomas / 中华放射学杂志

Objective:To investigate the MRI features of the primary sinonasal malignant melanoma (SMM) and evaluate the signal pattern based on T 1WI and T 2WI, in order to improve the diagnostic accuracy of SMM. Methods:The MRI findings of 63 SMM cases confirmed by pathology from April 2007 to November 2018 at Beijing Tongren Hospital, Capital Medical University were analyzed retrospectively. The signal intensity of malignant melanoma was classified into four types(Ⅰ—Ⅳ) according to the proportion of signal areas of the largest slice of the tumor on T 1WI and T 2WI. The classification criteria according to T 1WI: type Ⅰ, the area of hyperintensity was ≥50%; type Ⅱ, the area of hyperintensity was <50%; type Ⅲ, the tumor did not show hyperintensity, and the area of isointensity was ≥50%; type Ⅳ, the tumor did not have high signal area, and the area of low signal was ≥50%. The classification criteria according to T 2WI: type Ⅰ, the area of low signal in the tumor was ≥50%; type Ⅱ, the area of low signal was <50%; type Ⅲ, the tumor did not contain low signal area, and the area of isointensity was ≥50%; type Ⅳ, the tumor did not have low signal area, and the area of high signal intensity was ≥50%. The proportion of each type was calculated. Results:According to T 1WI, typeⅠwas identified in 27 cases (42.9%, 27/63), typeⅡ in 25 cases (39.7%, 25/63), type Ⅲ in 4 cases (6.3%, 4/63), and type Ⅳ in 7 cases (11.1%, 7/63). According to T 2WI, type Ⅰwas demonstrated in 29 cases (46.0%, 29/63), type Ⅱ in 28 cases (44.4%, 28/63), type Ⅲ in 2 cases (3.3%, 2/63), and type Ⅳ in 4 cases (6.3%, 4/63). There were 16 cases classified as type I based on T 1WI and T 2WI. Conclusions:Typical and atypical SMM can be identified according to signal patterns. The typeⅠsignal pattern of SMM cases on T 1WI and T 2WI is typical and can be easily diagnosed, but the proportion was less than 50%. For atypical SMM, malignant melanoma should be strongly suspected if hyperintense on T 1WI or hypointense on T 2WI is found.

Clinical characteristics and molecular genetics of seven neonates with congenital nephrogenic diabetes insipidus / 中华医学遗传学杂志

OBJECTIVE@#To explore the clinical characteristics, genetic basis and clinical treatment of seven neonates with congenital nephrogenic diabetes insipidus (NDI).@*METHODS@#Clinical data of the patients were collected. High-throughput sequencing was carried out to detect potential variants. Sanger sequencing was used to verify the results.@*RESULTS@#The patients were all males, with the age of onset being 10 to 21 days. All patients were admitted to the hospital for intermittent fever as the first symptom during the neonatal period. Additional symptoms had included polydipsia and polyuria. After the treatment, 5 patients had recovered, the remainders still had NDI symptoms and developmental retardation. Five children were found to harbor pathogenic variants of the AVPR2/AQP2 gene, which included one in-frame mutation of c.645_646insGCACCTACCCTGGGTATCGCC, two missense mutations of c.541C>T and c.419C>A, and two hemizygous deletions of the AVPR2/AQP2 gene. Among these, two were unreported previously. Cases 6 and 7 were a pair of twins. Both had carried homozygous missense variants of c.538G>A of the AVPR2/AQP2 gene, which was known to be pathogenic.@*CONCLUSION@#AVPR2/AQP2 is the main pathogenic gene for congenital NDI, for which two novel pathogenic variants have been discovered in this study. Above results have provided a basis for clinical diagnosis and genetic counseling for the affected pedigrees.

Analysis of NF1 gene variants among thirteen patients with neurofibromatosis type 1 / 中华医学遗传学杂志

OBJECTIVE@#To detect variants of NF1 gene among thirteen patients with neurofibromatosis type 1.@*METHODS@#Genomic DNA was extracted from peripheral blood samples of the patients. High-throughput sequencing was employed to detect potential variants of the NF1 and NF2 genes.@*RESULTS@#Thirteen pathogenic variants were identified among the patients, which included one NF1 deletion, three missense variants, three nonsense variants and six frameshifting variants. Among these, 10 variants have been associated with neurofibromatosis type 1. c.4180A>T (p.Asn1394Tyr), c.4217dupT (p.Leu1406fs) and c.1753dupT(p.Leu585Phefs*3) were unreported previously. Based on the guidelines of the American College of Medical Genetics and Genomics, c.4180A>T (p.Asn1394Tyr) was predicted to be likely pathogenic (PS2+PM1+PM2+PP2), while c.4217dupT (p.Leu1406fs) and c.1753dupT (p.Leu585Phefs*3) were predicted to be pathogenic (PVS1+PS2+PM2).@*CONCLUSION@#Variants of the NF1 gene probably underlay the disease among these children. Above findings have enriched the the spectrum of NF1 gene variants.

Clinical and genetic analysis of IL11RA-related Crouzon-like syndrome / 中华神经科杂志

Objective:To investigate the clinical manifestation, genetic characteristics, treatment and prognosis of Crouzon-like syndrome.Methods:Clinical data of one case of Crouzon-like syndrome diagnosed in Children′s Hospital Affiliated to Zhengzhou University in May 2019 were collected, including clinical test, treatment plan, follow-up outcomes. The clinical characteristics and the mutation characteristics of IL11RA-related Crouzon-like syndrome were analyzed combined with the literature.Results:The male proband, five years and four months old, was admitted with the main clinical manifestations including headache, vomiting, exophthalmos, ocular hypertelorism, nasal root flat and scaphocephaly. CT showed that the cerebellar tonsil moved down slightly, the occipital magnum was full, the bilateral cranial plates were locally thinner, the bilateral cranial diameters were increased, and the cranial seams were closed. Magnetic resonance imaging showed ChiariⅠmalformation. The mutation c.40_63del and splice site mutation c.811-2A>G of the patient′s IL11RA gene were screened by whole exome sequencing. Sanger sequencing showed that the mutations are compound heterozygous and both are first reported. The mutation c.811-2A>G was derived from the patient′s mother, and the other one is de novo.Conclusions:The main clinical manifestations of Crouzon-like syndrome are craniosynostosis and midface hypoplasia and ocular deformity. The study identified two novel mutations in the Crouzon-like syndrome related IL11RA gene. Genetic sequencing is helpful for accurate diagnosis and timely surgical treatment.

Investigation of microRNA-10a suppressing the activity of tumor-associated fibroblasts from a patient with metastatic colon cancer to liver / 中华老年医学杂志

Objective:To investigate the effects of microRNA-10a(miR-10a)on the proliferation and migration of tumor-associated fibroblasts(TAFs)in the liver microenvironment, as well as on the mRNA expressions of interleukin(IL)-6, IL-8 and IL-1β in TAFs.Methods:The normal liver tissues adjacent to cancer and focal tissues of metastatic colon cancer to the liver from the same patient were collected, and then primary normal fibroblasts(NFs)and the primary cell line of TAFs were established by tissue cultivation.The NFs and TAFs were identified by morphological observation and immunofluorescence staining, and their purity was determined by flow cytometry.The real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of miR-10a in NFs and TAFs, and then miR-10a was over-expressed in the lower ones.Subsequently, the effects of miR-10a on cell proliferation, migration and the mRNA expression levels of IL-6, IL-8 and IL-1β were detected by the cholecystokinin(CCK-8)test, wound healing assay and RT-qPCR.Results:Immunofluorescence staining showed that human cytokeratin 18(CK-18)was neither expressed in NFs nor in TAFs, while fibroblast-specific protein 1(FSP-1)was expressed in NFs and TAFs, and alpha-smooth muscle actin(α-SMA)was weakly expressed in NFs but strongly expressed in TAFs.The results of flow cytometry showed that the positive rates of α-SMA in NFs and TAFs were 95.6% and 95.3%, respectively.The mRNA expression of miR-10a in TAFs was 0.65 times of that in NFs( P<0.01). After overexpression of miR-10a, the proliferation abilities at the 3th, 4th and 5th day were lower in TAFs than in NFs( P<0.05 and 0.01), the migration abilities at 24 h and 48 h were 25% and 15% lower in TAFs than in NF group( P<0.01 and 0.05), and the mRNA levels of IL-6, IL-8, IL-1 β were 54%, 27% and 42% lower in TAFs than in NFs, respectively( P<0.01, 0.01 and 0.05). Conclusions:The overexpression of miR-10a in TAFs inhibits the cell proliferation and migration and reduces the mRNA expressions of inflammatory factors IL-6, IL-8 and IL-1β, which may be an important factor for TAFs’ inhibiting liver metastasis.

β-arrestin 1 facilitates acetylation of IL-6 gene promoter to affect proliferation and apoptosis of non-small cell lung cancer cells / 肿瘤

Objective: To investigate the effects of β-arrestin 1 (ARRB1) on apoptosis and proliferation of non-small cell lung cancer (NSCLC) cells, and to explore the underlying molecular mechanism. Methods: In NSCLC cell lines A549 and H1650, the expression of ARRB1 was knocked down by transfection of siRNA or over-expressed by transfection of Flag-ARRB1 recombinant plasmids, which was verified by real-time fluorescent quantitative PCR and Western blotting. The effects of down-regulating and up-regulating ARRB1 expression on the transcription and secretion of interleukin-6 (IL-6) were detected by real-time fluorescent quantitative PCR and ELISA method, respectively. The interaction between ARRB1 and p300 was detected by protein immunocoprecipitation. The recruitment of p300 and the acetylation of histone in IL-6 promoter region after ARRB1 knock-down or overexpression were detected by chromatin immunocoprecipitation. The proliferation and apoptosis of ARRB1 silencing A549 cells were detected by CCK-8 assay and FCM, respectively. The expression and activation of IL-6/signal transducer and activator of transcription 3 (STAT3) signaling pathway-related molecules in ARRB1 silencing or overexpression NSCLC cells were investigated by Western blotting. Results: In ARRB1-silenced or overexpressed NSCLC cell lines A549 and H1650, ARRB1 enhanced the transcription and production of IL-6 (all P < 0.05). The interaction of ARRB1 and p300 was confirmed by forward and reverse immunocoprecipitation. After ARRB1 knockdown or overexpression, it was found that ARRB1 enhanced the recruitment of p300 in IL-6 promoter region (both P < 0.01) and increased the acetylating of IL-6 promoter (both P < 0.05). Moreover, ARRB1 could facilitate the growth (P < 0.01) and apoptosis inhibition of NSCLC cells. ARRB1 could promote the phosphorylation of STAT3 and the expressions of c-Myc and Bcl-2 proteins. Conclusion: In NSCLC cells, ARRB1 interacts with p300, facilitates the recruitment of p300 to IL-6 promoter, and up-regulates the acetylation of histone H3 and H4 in IL-6 promoter, leading to transcriptional activation of IL-6. So that ARRB1 positively regulates the activation of IL-6/STAT3 signaling through promoting the phosphorylation of STAT3 and the expressions of c-Myc and Bcl-2 proteins, contributing to the growth and anti-apoptosis of NSCLC cells.

Application and Evaluation of Chemiluminescence Immunoassay in Blood Screening / 中国实验血液学杂志

OBJECTIVE@#To explore the application of blood screening method based on chemiluminescence immunoassay（CLIA）and to evaluate its officacy.@*METHODS@#Screening HBsAg, anti-HCV, anti-HIV and TP was performed on 3,530 voluntary blood donors by ELISA and CLIA, and then all the specimens with ELISA and ELISA/CLIA were further confirmed by NAT; TP single and double positive specimens by ELISA or CLIA were further confirmed by TPPA.@*RESULTS@#The results of CLIA method was well consistent with NAT results, displaying better repeatability and higher sensitivity than ELISA method. For CLIA/ELISA specimens there was a certain false-negative result obtained by ELISA method, especially for blood donors with low virus biter concentration or "window period".@*CONCLUSION@#ELISA and CLIA have complementary advantages in blood screening, which can improve the sensitivity of blood screening, reduce the missed detection and shorten detection time. The introduction of CLIA for blood screening is of great importance for ensuring the quality of blood and the safety of clinical transfusion.

Clinical characteristics and genetic analysis of primary coenzyme Q10 deficiency caused by COQ4 gene mutation / 中华内分泌代谢杂志

Objective@#To explore the clinical and genetic characteristics of primary coenzyme Q10 deficiency caused by coenzyme Q4 (COQ4) variants.@*Methods@#Clinical data were collected, while COQ4 gene was sequenced.@*Results@#Here were reported a boy of 3 months old who came to our hospital presented with feeding difficulties, repeated respiratory infections, convulsions for 3 months. He was subsequently diagnosed as cerebral atrophy, and growth retardation. All exons were sequenced.c.211G>A(p.A71T, maternal), c. 436T>A(p.F146I, paternal) were detected. After treatment with coenzyme Q10, the convulsive symptoms improved significantly. Literature review revealed that totally 14 cases with primary coenzyme Q10 deficiency caused by COQ4 gene mutation were reported. The onset age varies from neonatal to 18 years old, and the clinical manifestations are heterogeneous, including cardiomyopathy, epilepsy, ataxia, cerebellar atrophy, respiratory insufficiency, and growth retardation.@*Conclusion@#For cases with atypical clinical manifestations of primary coenzyme Q10 deficiency, gene detection is helpful for an early diagnosis and treatment.

The predictive value of right ventricular-pulmonary arterial coupling in patients with pulmonary arterial hypertension / 中国实用内科杂志

OBJECTIVE: To compare the predictive value of Ees/Ea in patients with pulmonary arterial hypertension based on right cardiac catheterization and cardiac magnetic resonance examination. METHODS: A total of 50 pulmonary arterial hypertension patients confirmed by right heart catheterization were retrospectively reviewed and followed up 2 years. The relationship between the baseline clinical data and survival prognosis of patients with pulmonary arterial hypertension were analyzed. The predictive value of baseline Ees/Ea for prognosis in patients with pulmonary arterial hypertension were compared. RESULTS: The Ees/Ea calculated by volume method was significantly correlated with the prognosis of patients with pulmonary arterial hypertension(OR =0.082, 95%CI 0.009-0.814, P=0.032). The two-year survival rate of group Ees/Ea>0.67 was significantly higher than that of Ees/Ea≤0.67(86.7% vs. 50%, P=0.003). Conclution Ees/Ea calculated by volume method is an independent influence factor of the survival prognosis of patients with pulmonary arterial hypertension. When Ees/Ea≤0.67, the patients with pulmonary arterial hypertension is more likely to deteriorate.

Clinical characteristics and genetic analysis of primary coenzyme Q10 deficiency caused by COQ4 gene mutation / 中华内分泌代谢杂志

Objective To explore the clinical and genetic characteristics of primary coenzyme Q10 deficiency caused by coenzyme Q4 ( COQ4) variants. Methods Clinical data were collected, while COQ4 gene was sequenced. Results Here were reported a boy of 3 months old who came to our hospital presented with feeding difficulties, repeated respiratory infections, convulsions for 3 months. He was subsequently diagnosed as cerebral atrophy, and growth retardation. All exons were sequenced.c.211G>A(p.A71T, maternal), c.436T>A(p.F146I, paternal) were detected. After treatment of coenzyme Q10, the convulsive symptoms improved significantly. Literature review revealed that totally 14 cases with primary coenzyme Q10 deficiency caused by COQ4 gene mutation were repoted. The onset age varies from neonatal to 18 years old, and the clinical manifestations are heterogeneous, including cardiomyopathy, epilepsy, ataxia, cerebellar atrophy, respiratory insufficiency, and growth retardation. Conclusion For cases with atypical clinical manifestations of primary coenzyme Q10 deficiency, gene detection is helpful for an early diagnosis and treatment.

Acceleration of wound healing by a porous collagen/silk fibroin scaffold carrying zinc oxide nanoparticles / 中国组织工程研究

BACKGROUND:Anti-infective ability determine the success or failure of skin grafting. It is one of the commonly used methods to enhance the anti-infective ability of implants by compounding antibacterial materials with scaffolds. OBJECTIVE:To investigate the effect of porous collagen/silk fibroin scaffolds carrying zinc oxide nanoparticles against infection and inflammation, and to evaluate its effect on wound healing. METHODS:Thirty-two Sprague-Dawley rats with a full-thickness wound on the back skin were randomly divided into two groups. In experimental groiup, porous collagen/silk fibroin scaffolds containing zinc oxide nanoparticles were implanted, while only collagen/silk fibroin scaffolds were implanted in control group. Wound healing was compared between the two groups by measuring residual wound area at 1, 2, 4, 8 weeks post implantation. Hematoxylin-eosin and interleukin 6 immumohistochemical staining were performed at 1, 2, 4 weeks post implantation to observe wound morphology and inflammatory reactions. Meanwhile, expression of interleukin 6 and interleukin 1β was detected by real-time PCR. RESULTS AND CONCLUSION:(1) At 2, 4, 8 weeks post implantation, significantly increased healing rate was observed in the experiment group compared with the control group (P<0.05). (2) Findings from the hematoxylin-eosin staining showed that obvious inflammatory cell infiltration was observed in the control group, but less inflammation with vigorous growth of granulation tissues on the wound surface occurred in the experimental group at 1 week after implantation. Then, the wound repair was basically completed in the experimental group presenting with complete and compact epidermal tissue structure, while scar formation with no skin cover was found in the control group at 4 weeks after implantation. (3) Findings from the interleukin 6 immumohistochemical staining showed that there was interleukin 6 positive expression in both two groups to different extents; at 4 weeks after implantation, the expression of interleukin 6 was remarkably reduced in the control group, but it was still a strong positive expression, while week positive expression of interleukin 6 was observed in the experimental group. (4) Compared with the control group, the mRNA expression of interleukin 6 and interleukin 1β was both lower in the experimental group at 1, 2, 4 weeks after implantation, but there was a significant difference between the two groups at 1 and 2 weeks after implantation (P<0.05). Overall, the porous collagen/silk fibroin scaffold carrying zinc oxide nanoparticles can effectively reduce inflammations following skin injury, and accelerate skin wound healing.