Effect of recombinant adenovirus Ad-mir-22 on glucose uptake in HepG2 cells / 生物工程学报

The recombinant adenoviruses expressing miR-22 (Ad-miR-22) was constructed and the effect of Ad-miR-22 on insulin signal pathway and glucose uptake in HepG2 cells was analyzed. MiR-22 gene was amplified by PCR from human hepatocytes and cloned into the pAdTrack-CMV vector to generate the shuttle plasmid pAdT-22. The positive colonies were confirmed by PCR and sequencing. The resultant shuttle plasmid was linearized with Pme I, followed by co-transformation into competent BJ5183 cells containing an adenoviral backbone plasmid (pAdEasy-1) to create the recombinant plasmid pAd-miR-22. After digested with Pac I, the linearized pAd-miR-22 was transfected into 293A packaging cell line to generate recombinant adenoviruses Ad-miR-22. HepG2 cells were infected with Ad-miR-22 or control Ad-GFP (adenoviruses expressing green fluorescent protein), and then the miR-22 expression levels were analyzed by qPCR. The result shows that adenovirus-mediated overexpression of miR-22 significantly decreased insulin-induced glucose uptake in HepG2 cells. Moreover, overexpression of miR-22 markedly decreased insulin-induced phosphorylation of GSK-3β. miR-22 also increased the mRNA levels of gluconeogenic genes in HepG2 cells. Furthermore, Western blotting results indicate that the protein expression of SIRT1 decreased in Ad-miR-22 infected HepG2 cells as compared with Ad-GFP infected HepG2 cells. In summary, overexpressing of miR-22 significantly increased gluconeogenesis while decreased glucose uptake in HepG2 cells. The effect of miR-22 on glucose metabolism may be mediated by SIRT1.

Clinical features and genetic analysis of intellectual disability in children with epilepsy of unknown causes / 中华实用儿科临床杂志

Objective To explore the clinical features,genetic causes and prognosis of intellectual disability with epilepsy(ID-E)in children.Methods The data of unknown causes of ID-E children(n=40)who were identified in Department of Pediatrics,Xiangya Hospital of Central South University from March 2015 to March 2016 were respectively analyzed,and follow-up studies were performed to investigate the epilepsy control and intellectual deve-lopment.Results Forty unexplained ID-E included 25(62.5％)male,and 34(85.0％)cases were severe intellectual disability patients.The onset age of epilepsy was 0.16 to 8.00 years old,median age was 1.5 years old.Twenty cases(50.0％)had slow electroencephalogram background,and 22 cases(55.0％)had focal spikes.Ten cases(25.0％)had abnormal cranial images,with brain dysplasia or atrophy.Follow-up lasted from 0.58 to 1.58 years,and 19 cases(47.5％)had seizure control.Twenty-five cases(62.5％)had used at least 2 anti-epilepsy drugs during follow-up,and 19 cases(47.5％)had drug refractory epilepsy.Improvement of mental or motor development in epilepsy controlled group and the uncontrolled group were 12 cases(63.2％)and 2 cases(9.5％).There were separately 8 cases(8/40 cases,20.0％)and 3 cases(3/16 cases,18.8％)diagnosed respectively by whole genome-wide analysis of copy number variants(CNVs)and gene-panel whose CNVs test findings were negative.Conclusions ID-E patients of unknown causes have the following clinical features:they were mostly found in male patients with severe intellectual disability,and drug refractory epilepsy patients have rather high percentage;well controlling of epilepsy is useful for improvement of mental and motor development.Genetic analysis is significant for control and prognosis of ID-E patients,and genome-wide CNVs have high positive rates which can be used as first-tier test to detect genetic etiology of ID-E of unknown cause.

Changes of febrile response and placental pathological inflammation in pregnant rats after intrauterine infection in late gestation / 中华围产医学杂志

Objective To explore the febrile response and placental pathological inflammation of pregnant rats exposed to intrauterine infection in late gestation.Methods Pregnant Sprague-Dawley rats at gestational day 18 were randomly divided into control group and intrauterine-infected group with six rats in each.The intrauterine-infected group was intraperitoneally injected with 350 μ g/kg lipopolysaccharide to establish a rat model of intrauterine infection,while the control group was injected with sterile saline of the same dose.Core temperature was measured every 1 h after intraperitoneal injection of lipopolysaccharide or saline for 8 h.At gestational day 19,after anesthesia,the placentas were taken and stained with HE.The expression levels of tumor necrosis factor-α,interleukin-6,and interleukin-1β in the placenta were determined by enzyme linked immunosorbent assay.Student t test was used for statistical analysis.Results (1) There was no temperature difference between the two groups before experimental treatment (P ＞ 0.05).Core temperature was increased 1 h after the lipopolysaccharide injection,reaching (37.67 ±0.08) ℃.The increase of temperature was significant compared with the control group [(37.13 ± 0.08) ℃,t=10.178,P ＜ 0.01].Fever was lowered 2 h later and the rats became hypothermic with body temperature below 37 ℃ in the intrauterine-infected group.The body temperature in the intrauterine-infected group after 2-6 h was (37.70 ± 0.10),(37.23 ± 0.05),(36.57 ± 0.06),(36.60 ± 0.10) and (36.57 ± 0.08) ℃,respectively,compared with the control group [(36.83 ±0.12),(36.63 ± 0.12),(36.71 ± 0.07),(36.87±0.12),and (36.77±0.08) ℃,respectively],the differences being all statistically significant (t=11.402,11.163,-4.025,-4.000 and-4.243,all P ＜ 0.01).(2) HE staining revealed large amounts of neutrophils infiltration,vascular enlargement and congestion in the placenta of the intrauterine-infected rats.No inflammatory cell infiltration was observed in the control placentas.(3) The expression levels of proinflammatory cytokine tumor necrosis factor-α [(0.62 ± 0.02) ng/g],interleukin-6 [(66.12 ± 5.11) ng/g],and interleukin-1β [(7.09± 1.23) ng/g] in the intrauterine-infected group were higher than those in the control group [(0.27±0.01),(16.71 ±1.55) and (2.86 ± 0.38) ng/g,respectively].The differences were all statistically significant (t=-26.608,-18.749 and-5.714,all P ＜ 0.01).Conclusion After exposure to lipopolysaccharide in late gestation,pregnant rats show significant inflammatory response in the placenta,with suppression of febrile response and presence of hypothermia.

Diagnostic value of head upright tilt testing on orthostatic hypotension in patients with senile hypertension / 中国医师进修杂志

Objective To evaluate diagnostic value of head upright tilt testing (HUTT) on orthostatic hypotension (OH) in patients with senile hypertension,and explore its efficacy and safety.Methods One hundred and sixty-six patients with senile hypertension were selected,and the patients were divided into 2 groups according to HUTT:experiment group (completed HUTT,86 cases) and control group (unfinished HUTT,80 cases).The blood pressure change was observed.The detection rate of OH and incidence of unexpected event were compared between 2 groups.Results The detection rate of OH in experiment group was 31.4％ (27/86),in control group was 17.5％ (14/80),and there was statistical difference (P ＜ 0.05).The incidence of unexpected event in experiment group was 0,in control group was 6.2％ (5/80),including 3 patients with dizziness amaurosis,unstable presyncope symptoms and fall,and 2 cases with syncope and sudden fall.There was statistical difference in incidence of unexpected event between 2 groups (P ＜ 0.05).Conclusions The use of HUTT in patients with senile hypertension can significantly improve the detection rate of OH,but also significantly reduce the incidence of unexpected event.The method is safe,effective,worthy of clinical promotion.

Analysis of a case with typical Hutchinson-Gilford progeria syndrome with scleroderma-like skin changes and review of literature / 中华儿科杂志

<p><b>OBJECTIVE</b>To explore clinical, radiographical and genetic characteristics of classical Hutchinson-Gilford progeria syndrome (HGPS).</p><p><b>METHOD</b>Data of a case of HGPS diagnosed at Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology was analyzed and related literature was reviewed.</p><p><b>RESULT</b>At the age of 8 months, the affected-infant presented with characteristic manifestation such as short stature, low weight, frontal bossing, alopecia, prominent scalp veins, micrognathia with a vertical midline groove in the chin, sclerodermatous skin, knee joints contracture with a horse-riding stance, and limited range of movement of ankle joints. Blood test showed blood platelet count (416-490) ×10(9)/L. Lower extremities MRI showed reduced subcutaneous fat. LMNA gene analysis showed that the affected-infant carried typical heterozygous mutation: c. 1824C>T (p. G608G), while his parents were normal. At the age of 13 months, X-rays showed short distal phalanges and clavicles with acro-osteolysis. After following up for 15 months, his appearance of progeria became more apparent. As far as we know, there are only 2 cases of classical HGPS confirmed by gene analysis in China.</p><p><b>CONCLUSION</b>Classical HGPS should be considered when infants appeared with sclerodermatous skin. Genetic analysis could help to diagnose classical HGPS as early as possible and avoid unnecessary investigations. In addition, affected-infants need to be long term followed-up and provided genetic counseling.</p>

The expressions of insulin receptor substrate in adipocytes during catch-up growth in neonatal rats with intrauterine growth retardation / 临床儿科杂志

Objective To investigate the expressions of insulin receptor substrate-1 (IRS-1) and insulin receptor sub-strate-2 (IRS-2) in adipocytes during catch-up growth in neonatal rats with intrauterine growth restriction (IUGR) and their correlations with the insulin resistance. Methods Sprague-Dawley rats (clean grade) were randomly divided into control group and food-restricted group after fertilization. Food-restricted group were received about 30%of food amount consumed in control group every day through the whole pregnant period to establish IUGR animal model, and were fed increased amount of breast-milk from postnatal day 1 to 21 to establish the period of catch-up growth in IUGR animal model (IUGR-CG). Fasting serum glu-cose, insulin and triglyceride were measured in blood from heart ventricles of 4-week old SD rats and insulin resistance index was calculated. Pre-adipocytes and mature adipocytes were obtained from SD rats at different age (1-week, 3-week, 5-week and 7-week old) and the former were induced to differentiate toward mature adipocytes. The levels of IRS-1, IRS-2 in the two kinds of mature adipocytes were detected by Real-Time PCR and Western blot. Results The expression levels of IRS-1, IRS-2 mRNA in mature adipocytes of IUGR-CG rats were signiifcantly lower than those of IUGR rats at 5-weeks and 7-weeks old (P<0.05) while the ex-pression levels of IRS-1, IRS-2 mRNA in differentiated adipocytes of IUGR-CG rats were signiifcantly lower than those of IUGR rats at 5-weeks old (P<0.05). The expression levels of IRS-1, IRS-2 protein in two kinds of adipocytes (mature and differentiated adipocytes) of IUGR-CG rats were signiifcantly lower than those of IUGR rats from postpartum week 1 through 7 (P<0.05). Conclusions IRS-1 and IRS-2 expression levels are downregulated in adipocytes during catch-up growth of IUGR rats, which may be closely related with insulin resistance.

Clinical and genetic analysis of two Chinese patients with isovaleric acidemia and review of literature / 中华实用儿科临床杂志

Objective To discuss the clinical features and treatment of isovaleric academia (IVA) patients,and to gain more comprehensive understanding of isovaleryl-CoA dehydrogenase(IVD) mutation in 2 siblings in order to raise awareness to prevent the occurrence of IVA.Methods The clinical history and laboratory test of 2 cases of children with IVA were carried out.The exons and neighboring introns of IVD gene of the whole family were PCR-amplified for DNA sequencing.The literature review of IVA in China was also conducted.Results Organic acid analysis of urine by GC/MS for both siblings showed extremely elevated concentrations of isovaleric glycine.For the older sibling,an acute episode of IVA caused severe metabolic stress and eventually death in the neonatal period.However,the disease was well-controlled for the younger sibling due to timely treatment and follow-up care for 2 years.The DNA sequencing of the IVD gene in the family revealed a novel c.1016G ＞ A(C339Y) heterozygous mutation in mother and both of the siblings.No IVD mutation was detected in father or in any of the 50 cases of healthy controls.According to literature review,15 cases of IVA were reported in recent 15 years in China,including neonatal onset (11 cases),acute episode (12 cases),odor of sweaty feet (12 cases),pancytopenia (9 cases),hyperammonemia (5 cases),hypocalcemia (6 cases),and 6 cases of death were reported.Additionally,5 cases that received treatment of BCAA-free formula milk showed positive outcome.However,only 2 cases were followed up for more than 2 years.Conclusions Two new IVA patients carrying c.1016G ＞ A(C339Y) mutation were reported in China.The mutation may lead to conformational change and functional deficient of the IVD protein.It is also necessary to point out that using direct DNA sequencing can not identify all patients with IVA due to limitations of this technology,and thus clinicians should be aware of the possibility of genetic misdiagnosis.Moreover,there is a trend of increasing IVA in China in recent years.

Empirical study for women and children institutions of infant mental health / 中华全科医师杂志

The samples of experimental and control groups were obtained by 1 ∶ 1 matching method and divided into development (242 pairs) and sign (156 pairs) groups.Through a 10-month group and case type intervention,Gesell test was performed for 5 zone development quotients of large motor,fine motor,adaptability,speech and personal-social to determinate the effectiveness of interventions.Before intervention,t values of the development group were 1.07,1.42,0.78,0.62 and 1.67 while those of sign group-1.48,-1.96,1.82,1.78 and-1.73 respectively.The inter-group comparison of various developmental quotient differences had no statistical significance (P ＞ 0.05).After intervention,t values of the development group were 9.85,8.92,3.57,11.21 and 9.30 for inter-group comparison and-31.65,-36.94,-46.26,-37.56 and-49.85 for intra-group comparison.The results of sign group were 10.91,9.61,10.75,12.01 and 14.36 for inter-group comparison and-23.20,-31.75,-44.94,-33.58 and-54.58 for intra-group comparison.Between intra-group and inter-group,the comparison of the developmental quotient differences had statistical significance (P ＜ 0.01).In two experimental groups,the post-intervention developmental quotients in 5 zones are better than those pre-intervention levels.It shows that preventive intervention of infant mental health is effective.