Objective To establish a real-time PCR based method for detection of survivin gene samples methylation with dye SYBR Green Ⅰ.Methods DNA samples from 25 pairs of gastric cancer tissue and matched normal gastric tissues were digested with mCpG-sensitive Hpa Ⅱand Msp Ⅰ, and detected by SYBR Green Ⅰ real-time PCR for survivin exon 1 specific primers. The concentration of digested DNA was monitored by real-time quantitative PCR with survivin intron 2 specific primers. A normal genomic DNA which served as a standard was diluted serially 10-fold to determine the sensitivity of real-time PCR. The products were further verified by agarose gel electrophoresis analysis.Results After HpaⅡ enzyme digestion, a peak of PCR product derived from methylated targeted gene was found in the melting curve at Tm (91.5±0.5) ℃, which was verified as 338 bp strap. The control gene (survivin intron 2) characteristic of Tm (79.5±0.5) ℃ was amplified in all digested samples, indicating no non-specific degradation occurred in the genome DNA. The sensitivity of this method was 100 copies/μL. The new established PCR system was applied to detecting 25 cases of lung cancer tissue samples. As a result, the frequency of survivin exon 1 demethylation was 96%.Conclusion As a reliable new method for detection of survivin exon 1 methylation, SYBR Green Ⅰ real-time fluorescent PCR assay is rapid, accurate, sensitive, timing and simple.

Survivin variants specific real time quantitative RT-PCR was developed to analyze their expression in 53 paired cancer and para-cancerous tissues, and the expression of the wild-type survivin protein was detected by immunohistochemistry. The results showed that survivin mRNA and protein were expressed in gastric cancer and para-cancerous tissues. The survivin-2B was dominantly expressed in para-cancerous tissues, whereas the survivin-DeltaEx3 was more frequently detected in cancer tissues. The positive rate of survivin-2a was 100% in both cancer and para-cancerous tissues, but its relative transcript expression level was not significantly increased in cancer tissues in comparison with para-cancerous tissues. The correlation analysis revealed that the expression of survivin-2a mRNA was significantly associated with that of total survivin (r (s)=0.4178, P=0.0018), whereas inversely to that of survivin-DeltaEX3 (r (s)=-0.4506, P=0.0007). It was suggested that survivin-2a may act as an antagonist of survivin-DeltaEX3. The balance between antiapoptotic survivin iso-forms and nonantiapoptotic ones may play an important role in tumorigenesis and tumor progression. Promising value is hinted to analyze survivin and its variants in tumor early diagnosis and distinguishing malignant tumors from benign ones.
Microtubule-Associated Proteins/genetics ; Microtubule-Associated Proteins/*metabolism ; Protein Isoforms/genetics ; Protein Isoforms/metabolism ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; Stomach Neoplasms/*metabolism ; Stomach Neoplasms/pathology ; Tumor Cells, Cultured ; Tumor Markers, Biological/genetics ; Tumor Markers, Biological/*metabolism

Objective To investigate the correlations of expression of interferon inducible protein 10 ( IP-10) mRNA in peripheral blood mononuclear cells ( PBMCs) with serum levels of HBsAg and HBV DNA in patients with HBV-related acute on chronic liver failure ( HBV-ACLF ) , and to assess its value in predicting disease prognosis .Methods Eighty patients with HBV-ACLF, 60 patients with chronic hepatitis B (CHB), and 25 healthy subjects were enrolled during October 2013 and February 2015.IP-10 mRNA in PBMCs was measured by real time quantitative polymerase chain reaction ( RT-qPCR) , and the difference in IP-10 mRNA expression among three groups was compared by ANOVA .Serum levels of HBsAg and HBV DNA were also measured in patients with HBV-ACLF, and Pearson correlation test was performed to analyze the correlations of IP-10 mRNA with HBsAg and HBV DNA levels .t test was used to analyze the differences in IP-10 mRNA, HBsAg and HBV DNA levels between fatal and surviving cases after 3-month entecavir therapy in HBV-ACLF group.Receiver operating characteristic curves ( ROC) was used to evaluate the prognostic value of IP-10 mRNA, HBsAg and HBV DNA in HBV-ACLF patients .Results IP-10 mRNA level in HBV-ACLF group was 0.998 ±0.186, which was higher than those in CHB patients and healthy controls (0.641 ±0.083 and 0.412 ±0.062, t=3.841 and 16.661, P<0.01).Pearson correlation analysis showed that IP-10 mRNA level was negatively correlated with HBsAg level in HBV-ACLF patients (r=-0.576, P<0.01), but positively correlated with HBV DNA level (r=0.547, P<0.01).After 3-month entecavir therapy , IP-10 mRNA level in surviving cases of HBV-ACLF group was 0.894 ±0.164, which was lower than that in fatal cases ( 1.103 ±0.177, t =-4.328, P <0.01 ); HBsAg level in surviving cases was higher than that in fatal cases (3.303 ±0.565 vs.2.605 ±0.844, t =3.251, P<0.01).The area under ROC of IP-10 mRNA in evaluating prognosis of HBV-ACLF was 0.820, which was higher than those of HBsAg (0.663) and HBV DNA (0.570).Conclusions IP-10 mRNA in patients with HBV-ACLF is over-expressed and is correlated with HBsAg and HBV DNA levels .It may be used for predicting the prognosis of patients with HBV-ACLF.

Objective To explore the effects of morroniside on the expression of CD34 in ipsilateral cortex of rats after focal cerebral isch-emia-reperfusion. Methods 45 male Sprague-Dawley rats were divided into sham group (n=9), ischemia group (n=9), and morroniside groups (low, medium and high dosage groups, n=9). The middle cerebral artery were occluded for 30 minutes, and reperfused. Morroniside was administered intragastrically once a day at dose of 30 mg/kg, 90 mg/kg, 270 mg/kg after operation. The expression of CD34 in the isch-emic ipsilateral cortex were detected with immunohistochemistry (n=6) and Western blotting (n=3) 7 days after operation. Results The ex-pression of CD34 increased in the ischemia group compared with the sham group, and further increased in the morroniside groups of high dos-age compared with the ischemia group (F>14.865, P<0.001). Conclusion Morroniside could increase the expression of CD34 in the ischemic ipsilateral cortex after ischemia-reperfusion in rats, which may promote the angiogenesis and neurogenesis after ischemia.

Objective To investigate the effects of morroniside on the expression of vascular endothelial growth factor (VEGF) and fi-broblast growth factor-2 (FGF-2) in rat cortex after focal cerebral ischemia-reperfusion. Methods 30 male Sprague-Dawley rats were ran-domly divided into sham group, model group, morroniside-low group (30 mg/kg), morroniside-middle group (90 mg/kg) and morroni-side-high group (270 mg/kg). Middle cerebral arteries of rats were occluded for 30 minutes with Longa's method and re-perfused. The ex-pression of VEGF and FGF-2 in the ischemic ipsilateral cortex was detected with Western blotting 7 days after reperfusion. Results The ex-pression of both VEGF and FGF-2 increased in the ischemic ipsilateral cortexin in all the ischemic groups compared with the sham group (P<0.05). The expression of VEGF further increased in a dose-dependent manner in all the morroniside groups compared with that of model group (P<0.05), and the expression of FGF-2 increased in the morroniside-high group (P<0.001). Conclusion Morroniside could increase the expression of VEGF and FGF-2 after ischemia-reperfusion, which might promote angiogenesis.

Survivin variants specific real time quantitative RT-PCR was developed to analyze their expression in 53 paired cancer and para-cancerous tissues, and the expression of the wild-type survivin protein was detected by immunohistochemistry. The results showed that survivin mRNA and protein were expressed in gastric cancer and para-cancerous tissues. The survivin-2B was dominantly expressed in para-cancerous tissues, whereas the survivin-△Ex3 was more frequently detected in cancer tissues. The positive rate of survivin-2a was 100% in both cancer and para-cancerous tissues,but its relative transcript expression level was not significantly increased in cancer tissues in comparison with para-cancerous tissues. The correlation analysis revealed that the expression of survivin-2a mRNA was significantly associated with that of total survivin (rs=0.4178, P=0.0018),whereas inversely to that of survivin-/EX3 (rs=-0.4506, P=0.0007). It was suggested that survivin-2a may act as an antagonist of survivin-△EX3. The balance between antiapoptotic survivin iso-forms and nonantiapoptotic ones may play an important role in tumorigenesis and tumor progression. Promising value is hinted to analyze survivin and its variants in tumor early diagnosis and distinguishing malignant tumors from benign ones.

Objective:To summarize the clinical phenotypes and genetic variations of children with epilepsy related to CLCN4 gene mutations. Methods:A retrospective analysis was conducted on 9 children with epilepsy who were diagnosed with CLCN4 gene mutations through whole-exome sequencing of family members. These children were treated at the Department of Pediatrics, Peking University First Hospital from December 2016 to March 2024. Their clinical manifestations, electroencephalogram, cranial imaging characteristics, and treatment follow-up were reviewed. Results:Among the 9 children, 6 were male and 3 were female. All cases involved de novo mutations. Three cases carried the c.823G>A/p.V275M variant, 2 cases carried the c.2152C>T/ p.R718W variant, 1 case carried the c.1630G>A/pG544R variant, and 1 case carried the c.2167C>T/ p.R723W variant. Two cases carried the unreported new variant c.848G>T/p.S283I and c.818G>A/ p.G273E. The onset age of epilepsy ranged from 55 days to 10 years, with a median onset age of 14 months. Seven out of 9 children had epilepsy onset before the age of 2 years. The types of seizures varied: 8 had focal seizures, 1 had generalized tonic-clonic seizures, 2 had myoclonic seizures, 1 had epileptic spasms, and 1 had atypical absence seizures. Three children experienced multiple types of seizures. All 9 children exhibited developmental delays to varying degrees: 8 had global developmental delay and 1 had cognitive developmental delay. Developmental delays were observed in 7 children before the onset of epilepsy. Clinically, 1 child was diagnosed with infantile epileptic spasms syndrome, 7 with unclassified developmental and epileptic encephalopathy, and 1 with focal epilepsy with developmental delay. At the last follow-up, the age of the children ranged from 2 years and 5 months to 13 years and 9 months. Seizures had been controlled in 3 children for a duration of 4 to 12 months. Conclusions:De novo variants are common in CLCN4 variants. Most seizures onset in infancy, seizure types are various, and focal seizures are common. Most of them have developmental delay and drug-resistant epilepsy, and some of them have developmental delay before seizure onset, which is consistent with the characteristics of developmental and epileptic encephalopathy.

The study was a retrospective study. The clinical data of 866 patients with IgA nephropathy (IgAN) in Beijing Anzhen Hospital, Capital Medical University from March 2010 to March 2021 were analyzed, to investigate the clinical pathology and renal prognosis of IgAN patients with intrarenal arteriolosclerosis, and to preliminarily explore whether abnormal activation of complement system is involved in the injury of arteriolosclerosis. The patients were divided into renal arteriolar lesions group and non-renal arteriolar lesions group according to the renal histopathology, and the differences of clinical pathological manifestations, prognosis between the two groups were compared. The results showed that, compared with the non-renal arteriolar lesions group ( n=236), IgAN patients in the renal arteriolar lesions group ( n=630) had higher proportions of hypertension and malignant hypertension, higher levels of urinary albumin-creatinine ratio, 24-hour urine protein quantification and serum uric acid, lower estimated glomerular filtration rate, and more severe MEST-C lesions of the Oxford classification (all P < 0.05). Cox regression analysis results showed that intrarenal arteriolosclerosis was the independent risk factor affecting the progression of IgAN to ESRD ( HR=6.437, 95% CI 2.013-20.585, P=0.002). Renal histopathology showed that the deposition of complement C3c on the wall of intrarenal arterioles in the renal arteriolar lesions group ( n=98) was stronger than that in non-renal arteriolar lesions group ( n=18, P < 0.05). IgAN patients with renal arteriolosclerosis present with serious clinical and pathological manifestations, and renal prognosis. Abnormal activation of complement system may be involved in the pathogenesis of intrarenal arteriolosclerosis.

Objective To investigate whether the clinical and pathological injury of kidney in IgA nephropathy (IgAN) patients with hypertension is associated with circadian blood pressure rhythm change, particularly with elevated nocturnal blood pressure (BP). Methods This study was a retrospective cross-sectional study. Clinic and renal histopathological injury data were obtained from 83 IgAN patients with hypertension. First, 24 h ambulatory BP monitoring (ABPM) data were analyzed. Second, all these IgAN patients were divided into two groups, elevated nocturnal BP group and nocturnal normotensive BP group, and the clinical and pathological differences between this two groups were analyzed. Third, logistic regression analysis was used to analyze the influencing factors of renal tubulointerstitial injury in IgAN patients with hypertension. At last, all these IgAN patients were divided into two groups according to the level of estimated glomerular filtration rate (eGFR), group of patients with eGFR≥60 ml·min-1·(1.73 m2)-1 and the other group with eGFR<60 ml·min-1·(1.73 m2)-1, and the 24 h ABPM data were compared. Results (1) The proportion of non-dipper circadian rhythm of BP in IgAN patients with hypertension was 79.5％. (2) Compared with nocturnal normotensive BP group, patients in elevated nocturnal BP group had significantly higher levels of 24-hour urinary protein quantity and blood uric acid (both P<0.05), and lower eGFR and urine osmotic pressure clinically (both P<0.05). Index of interstitial fibrosis and tubular atrophy was significantly higher in nocturnal normotensive BP group (P<0.05), while the proportion of glomerular ischemia lesion was not significantly different between two groups. (3) Multivariate logistic regression analysis showed that elevated nocturnal BP was an independent risk factor for severe tubulointerstitial injury of IgAN (OR=1.113, 95％CI 1.038-1.192, P=0.002). (4) Compared with the group of eGFR≥60 ml·min-1·(1.73 m2)-1, 24-hour systolic blood pressure (SBP) and diastolic blood pressure (DBP), daytime SBP and DBP, nocturnal SBP and DBP were significantly higher in group of eGFR<60 ml·min-1·(1.73 m2)-1 (all P<0.05). Conclusion The proportion of non-dipper circadian rhythm of BP in IgAN patients with hypertension is as high as 79.5％. Elevated nocturnal BP is associated with the severity of renal damage, and elevated nocturnal BP is an independent risk factor for severe tubulointerstitial injury in IgAN patients with hypertension. Therefore, 24 h ABPM should be emphasized, and elevated nocturnal BP should be well controlled to slow the progression of IgAN.

Methods:Whole blood and serum samples were collected from 86 patients with HBV-ACLF and 42 patients with chronic hepatitis B (CHB). Serum alanine aminotransferase (ALT), total bilirubin (TBil), creatinine (Cr), albumin (ALB) cholinesterase (CHE) and international standardized ratio (INR) were detected by routine method . The CXCL10 mRNA level of peripheral blood mononuclear cells was detected by fluorescence quantitative PCR. The genotype and allele of rs56061981 were detected by direct DNA sequencing. The genotype and allele frequencies between the ACLF and CHB groups were compared. The levels of ALT, TBil, INR, ALB, CHE, model for end-stage liver disease (MELD) and CXCL10 mRNA were compared between the two groups according to whether the T allele was carried.Results:There was no significant difference in sex, age, alcohol history, smoking history, HBV genotype, HBeAg status and HBV-DNA level between CHB group and HBV-ACLF group (P > 0.05). The frequency of CT+ TT genotype and the frequency of T allele at the rs56061981 locus of the CXCL10 gene in group ACLF were significantly higher than that in the CHB group ( χ2=4.83, P=0.03 and χ2=4.95, P=0.03). The difference is significant Genotype CT+ TT is a risk factor for CHB to develop into HBV-ACLF ( OR=2.897, 95% CI: 1.09~7.68). Allele T is a risk factor for CHB to develop into HBV-ACLF ( OR=2.746, 95% CI: 1.10~6.89). The plasma INR and MELD scores of ACLF individuals carrying T alleles were significantly higher than those carrying C alleles (t=2.63, P=0.013 and t=2.7, P=0.011). The difference is significant. Albumin (ALB) and cholinesterase (CHE) was significantly lower than those carrying C alleles (t=2.67, P=0.01 and t=3.545, P=0.001). The difference is significant. But there was no significant difference in serum TBL and ALT between the two groups. Conclusions:The polymorphism of CXCL10 gene rs56061981 is significantly correlated with the susceptibility and severity of ACLF. Allele T may be a susceptible gene of ACLF, and it can also be used as a predictor of the severity of the disease.