Objective To make molecular diagnosis for puerile spinal muscular atrophy(SMA).Methods Genomic DNA was extracted directly from the blood of both the case group(10 children with SMA) and the control group(including 19 parents of SMA patients and 20 healthy individuals).Two methods,polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and allele-specific PCR,were used to analyze exon 7 of SMN gene from genomic DNA,and consequent electrophoresis of PCR products on agarose gel was performed.Results Genotyping results obtained by both methods were in complete agreement for all of the samples analyzed.In conventional PCR-RFLP,part of the PCR products(189bp) from genomic DNA of all 39 members in the control group remained intact after digestion with Dra I,while the PCR products from genomic DNA of all 10 SMA children in the case group was completely digested by Dra I.In allele-specific PCR,exon 7 of both SMN1 and SMN2 could be seen when genomic DNA of all 39 members in the control group was used,while only SMN2's exon 7 could be seen when genomic DNA of all 10 SMA children in the case group was used.Conclusion Homozygous deletion of SMN1 was present in all 10 SMA children in the case group,while homozygous deletion of SMN1 was not detected in all 39 members in the control group.The combination of PCR-RFLP and allele-specific PCR,both their results can be references for each other,offers efficient and accurate methodology for molecular diagnosis of SMA.

In recent years , active vitamin D is a hot research drugs because of its renal protective effect of independent regu -lation of anti-inflammatory effects outside calcium and phosphorus metabolism , regulation of apoptosis , mediated immunity and reduc-tion of proteinuria .Podocyte is the main target of active vitamin D based on the result of clinical and animal studies .In this article, we review the current literature on mechanism of active vitamin D and its analogues on the protection of podocytes about and give the clini -cal perspectives of activity vitamin D .

Objective To perform fetal karyotyping by percutaneous umbilical blood sampling guided by color Doppler ultrasonography in fetuses with congenital cardiac anomalies. Methods Fetal blood samples obtained by color Doppler ultrasound-guided cordocentesis in 56 fetuses with cardiac abnormalities detected by fetal echocardiography were taken for karyotyping.Fetuses were monitored post operation. Results The procedures were successfully performed in all cases and no procedure-related complications occurred.Six cases with abnormal karyotypes, 1 trisomy 21,2 trisomy 18,1 trisomy 13,1 trisomy AO and 1 cases 47XYY were identified and pre-operation ultrasonography detected multiple system anomalies besides cardiac anomalies in them. Conclusions Color Doppler ultrasonography facilitated percutaneous umbilical blood sampling by clear umbilical vein imaging and shortening the operation time.Abnormal karyotypes in fetuses with cardiac anomalies seemed to be related with multiple system anomalies.

Objective The core mechanism of renal insterstitial fibrosis (RIF) is epithelial-mesenchymal transition.This study aimed to investigate the effect of erythropoietin on high glucose-induced epithelial-mesenchymal transition ( EMT) of normal hu-man kidney proximal tubular epithelial cells (HK-2) and its possible mechanism. Mothods HK-2 cells cultured in vitro were ran-domly divided into a blank control group , a high glucose induction group , a mannitol induction group , an EPO induction group , an EPO (5, 10, and 20U/mL) inhibition group, and an Rho kinase inhibitor group.After 24 hours of intervention, the mRNA levels of RhoA and ROCK were determined by RT-PCR, those of E-cadherin and α-smooth muscle actin (α-SMA) proteins detected by immu-nofluorescence staining , and the expression of FN proteins in the supernatant measured by ELISA . Results Compared with the blank control group , the expressions of RhoA and ROCK 1 mRNA were significantly increased in the high glucose induction group (0.945 ±0.132 vs 1.400 ±0.022, 1.007 ±0.002 vs 1.913 ±0.011, P<0.05), but markedly decreased in the 5, 10, and 20U/mL EPO inhibition groups (1.400 ±0.022 vs 1.278 ±0.006, 1.400 ±0.022 vs 0.770 ±0.005, 1.400 ±0.022 vs 0.334 ±0.009, P<0.006) in comparison with the high glucose induction group , and the effects were related to the concentration of EPO .Compared with the blank control, the expression of E-cadherin protein was increased in the high glucose induction group (0.644 ±0.006 vs 0.107 ± 0.004, P<0.05), but remarkably decreased in the 5, 10, and 20 U/mL EPO inhibition groups (0.236 ±0.006, 0.433 ±0.010, 0.521 ±0.010) in comparison with the high glucose induction group (P<0.05), and the effects were also related to the concentration of EPO.Pearson correlation analysis showed a positive correlation between the mRNA expressions of RhoA and ROCK 1 in the high glu-cose induction and EPO inhibition groups . Conclusion EPO can inhibit high glucose-induced epithelial-mesenchymal transition of normal human kidney HK-2 cells and thus delay renal fibrosis , which mignt be related to the RhoA/ROCK signaling pathway .

Objective To construct a recombinant vector of sperm-specific human lactate dehydrogenase ( hLDH-C4 ), express it in Escherichia coli ( E. coli ) BL21 ( DE3 ) and utilize it in the detection of anti-sperm antibody. Methods The coding sequence of hLDH-C4 was amplified from human testis λTripIEx cDNA library, and inserted into pET-28a( + ) after restriction enzyme digestion with Hind Ⅲ and Xho Ⅰ. The resultant recombinant vector was used to transform E. coli BL21 ( DE3 ) and the His-Tag fused hLDH-C4 was expressed after induction with IPTG. Western blot was used to analyzed the recombinant protein and LDH activity of bacterial lysates was determined. An indirect ELISA method for the detection of anti-sperm antibody was established by using the recombinant hLDH-C4 as antigen matrix. Results pET-28a( + )-hLDHC was successfully established. The protein with size of 35kD could be induced by IPTG when the recombinant plasmid was transfected into E. coli BL21 ( DE3 ). Western blot showed that the recombinant protein could be specifically recognized beth by anti-His tag monoclonal antibody and by rabbit anti-human LDH-C4 antibody. In addition, the recombinant protein showed high-level LDH activity when the bacterial lysate after IPIG induction was used to check LDH activity. The recombinant hLDH-C4 was confirmed when it was used in indirect EL1SA to detect anti-hLDH-C4 antibody. Conclusions The coding sequence of hLDH-C4 is cloned into the vector pET-28a( + ) and recombinant hLDH-C4 was expressed at a high level in E. coli. The recombinant hLDH-C4 is useful in the detection of anti-sperm antibody.

Objective Clinical treatment can delay the development of renal interstitial fibrosis , but it can not reverse renal dysfuntion.The article was to discuss the influence of recombinant human erythropoietin ( rHuEPO ) on inflammatory factors in the process of renal interstitial fibrosis and its possible mechanism . Methods The vitro cultured HK-2 cells were randomized into 7 groups:the blank control group , rHuEPO control group ( addition of 20U/mL rHuEPO), albumin stimulation group (addition of 5mg/mL albumin), 5mg/mL rHuEPO intervention group (5mg/mL albumin +5U/mL rHuEPO), 10 U/mL rHuEPO intervention group (5mg/mL albumin +10 U/mL rHuEPO), 20U/mL rHuEPO intervention group (5mg/mL albumin +20U/mL rHuEPO), and Rho inhibi-taion group (addition of 5mg/mL albumin 30min after 10μmol/L Y27632), 24 h acting time for each group.We observed the changes of cell morphology in each group .Reverse transcription polymerase chain reaction ( RT-PCR) was used to evaluate the mRNA levels of RhoA, ROCK1 and IL-6 , and ELISA was applied to measure the levels of supernatant TNF-αand IL-6 protein. Results The form of pebbles or paving stone was observed in blank control group and rHuEPO intervention groups , a long and thin spindle change with the appearance of fibre cells in albumin stimulation group , the transformation to pebbles in 5, 10, 20 mg/mL rHuEPO intervention groups , the form of oval and slightly increased intercellular space in Rho inhibitaion group .Compared with the blank control group , the expressions of RhoA mRNA, ROCK1 mRNA and IL-6 mRNA significantly increased in the albumin stimulation group (P<0.05), while significantly reduced in 5, 10, 20 mg/mL rHuEPO intervention groups (P<0.05), which was in negative relation with the rHuEPO concentrations .Compared with the albumin stimulation group , the expressions of ROCK 1 mRNA and IL-6 mRNA reduced in Rho inhibtation group (P<0.05), while there was no significant difference as to the expression of RhoA mRNA .ELISA results showed:compared with blank control group , the expressions of supernatant TNF-α([452.32 ±33.23] ng/L vs [1347.54 ±41.52] ng/L), IL-6 protein([884.62 ±0.73] pg/L vs [95.12 ±0.32]pg/LP<0.05) increased significantly.Compared with albumin stim-ulation group, the expressions of TNF-αin 5, 10, 20 mg/mL rHuEPO intervention groups and Rho inhibitation group reduced signifi-cantly([1003.32 ±3.42] ng/L, [821.32 ±21.32] ng/L, [590.15 ±7.68] ng/L, [488.13 ±65.03] ng/L vs [1 347.54 ± 41.52]ng/L,P<0.05), while the expressions of IL-6 mRNA reduced accordingly in 5, 10, 20 mg/mL rHuEPO intervention groups and Rho inhibitation group reduced significantly ([656.68 ±0.55] pg/L, [422.35 ±0.22] pg/L, [217.32 ±0.35] pg/L, [309.49 ±0.21] pg/L vs [884.62 ±0.73]pg/L,P<0.05).Moreover, there was significant statistical difference among 5, 10, 20 mg/mL rHuEPO intervention groups(P<0.05). Conclusion RHuEPO can inhibit the transdifferentiation process of HK-2 cells in-duced by albumin by suppressing inflammation factors , and the mechanism may be involved in RhoA/ROCK signaling pathway .

Objective To investigate methods for prenatal molecular diagnosis of fetuses at high risk for X-linked adrenoleukodystrophy(X-ALD).Methods The amniotic fluid was obtained and genomic DNA was isolated from amniotic fluid cells.Maternal contamination was evaluated by paternity test.PCRRFLP,sequencing and denaturing high performance liquid chromatography(DHPLC)were used to detect the ABCD1 gene of fetal genome.Results In the pedigree 1,the PCR product(799 bp)of the fetus 1 and her father(normal control)could be digested with BcnI. No P560L mutation,which was present in the index patient,was detected in the ABCD1 gene from the genomic DNA of the fetus 1 using direct sequencing.In the pedigree 2,the PCR product(232 bp)of the fetus 2 and her father could not be digested with MaeI and no Q177X mutation,which was present in the propositus,was detected in the ABCD1 gene from the genomic DNA of the fetus 2 using direct sequencing.In the pedigree 3,the PCR product(271 bp)was digested with AciI.the pattern of the fetus 3 and the propositus being the same,and the R617C mutation was found in the ABCD1 gene from the genomic DNA of the fetus 3 using direct sequencing.In the pedigree 4,the PCR product(269 bp)was analyzed with the DHPLC,and the pattern of elution peaks of the fetus 4 and her father was similar,but different from that of the propositus.No K276E mutation was detectable in the ABCD1 gene from the genomic DNA of the fetus 4 by using direct sequencing.Judging from the sex of the fetuses,fetuses 1 and 2 were normal homozygotes,fetus 3 was an ALD hemizygote,and fetus 4 was a normal hemizygote.Conclusion A new protocol for X-ALD prenatal molecular diagnosis is proposed,which would ensure the accuracy of prenatal diagnosis.

Objective To study the effects of erythropoietin (rhEPO) in high glucose induced proliferation and apopto?sis of human kidney proximal tubular epithelial (HK-2) cells, and the possible mechanism thereof. Methods HK-2 cells cultured in vitro were divided into several groups randomly:blank control group, high glucose group, mannitol group, rhEPO control group, different concentrations of rhEPO treatment groups (5, 10, 20 U/mL) and Rho kinase group. The reverse tran?scription polymerase chain reaction (RT-PCR) was used to evaluate the mRNA levels of RhoA and ROCK after 24 hours. Tetrazolium salt method (MTT) was used to determine the cell proliferation. Cell apoptosis was detected by flow cytometry. Results Compared with blank control group the expression levels of RhoA and ROCK1 mRNA were significantly in?creased in high glucose group (P < 0.05). RhoA, ROCK1 mRNA expressions significantly decreased in rhEPO group than those of high glucose group (P<0.05). There was a positive correlation between the expression levels of RhoA mRNA and ROCK1 mRNA in high glucose group and rhEPO group. MTT method showed that rhEPO significantly promoted the prolifer?ation of HK-2 cells (P<0.05). Flow cytometry analysis showed that high glucose induced apoptosis in HK-2 cells, which was significantly inhibited in rhEPO group and Rho kinase group as compared to that of high glucose group in a concentra?tion dependent manner (P<0.05). Conclusion rhEPO can promote HK-2 cell proliferation and inhibit apoptosis, which may be related to RhoA/ROCK signaling pathway.

Objective To study the correlation between the diffusion-weighted imaging (DWI) measurements and glomerular filtration rate (GFR), Katafuchi scores in IgA nephropathy. Methods Thirty-five patients with IgA nephropathy (IgAN group) and twenty healthy volunteers (control group) were enrolled in this study. All of the subjects underwent bilateral renal DWI measurements with 3.0T MRI scanner. The values of apparent diffusion coefficient (ADC) of renal cortex and medulla were measured. GFR of IgAN group was detected with 99Tcm-DTPA scintigraphy. Based on the Lee classification and the Katafuchi score system, the pathological grading was carried out in patients of IgAN group. The ADC values were compared between control group and different grades of IgAN group. The correlations between ADC and GFR values were analysed in defferent groups. The correlations between ADC values and Katafuchi scores were analysed in IgAN group. Results The renal cortical ADC values were significantly higher than medulla ADC values in both control group and IgAN group (P < 0.05). There were statistically significant differences in renal cortical ADC values and medulla ADC values between control group and IgAN subgroups (P<0.05). But there was no significant difference in renal cortical ADC value between IgANⅠgroup and control group (P>0.05). There was a positive correlation between the renal cortical and medulla ADC values and the GFR values in IgAN group (P<0.01). Negative correlation was found between the renal cortical and
medulla ADC values and the Katafuchi scores in IgAN group (P<0.05). Conclusion The diffusion-weighted imaging can reflect the physiological functions of kidney. It was feasible for application DWI in IgA nephropathy, which can be used for assessing the renal filtration function and the pathological damage. However, DWI measurement is not sensitive to early renal disease.

OBJECTIVETo explore the source of small supernumerary marker chromosome in a case.

METHODSG-banded karyotyping, fluorescence in situ hybridization, multiple sequence tagged sites (STS) of the Y chromosome, and Illumima Human Cyto SNP-12 Beadchip analysis were carried out.

RESULTSThe karyotype was mos 46,X,+mar1[21]/46,X,+mar2[78]. Y chromosome STS analysis has displayed the presence of sy84, sY86, USP9Y and DDX3Y genes from the AZFa region, and sY1227 of the AZFb region, while sY1228, sY1015, sY127, sY134 from the AZFb region, and sY254 and sY255 from the AZFc region were missing. FISH analysis has verified both of the marker chromosomes to be Y chromosome fragments. Mar1 was ish.idic(Y)(q11.2)(SRY++,DXZ1+,DYZ3++,DYZ1-), while mar2 was ish.del(Y)(q11.2)(SRY+,DXZ1+,DYZ3+,DYZ1-). Single nucleotide polymorphism (SNP) microarray analysis showed that the Yq11.2-Yq12 has lost a 10.81 Mb fragment.

CONCLUSIONThe marker chromosomes were verified to be aberrant Y chromosomes, with the breakage and recombination occurring in Yq11.2. Mar 1 was an isodicentric Y chromosome (idic(Y)pter to q11.2::q11.2 to pter), and mar2 was del(Y)(q11.2). The karyotype was mos 46,X,ish idic(Y)(q11.2)(DYZ3++,SRY++,DXZ1+,DYZ1-)[21]/46,X,ish del(Y)(q11.2)(DYZ3+,SRY+,DXZ1+,DYZ1-)[78]. Combined FISH, Y chromosome STS analysis, SNP microarray analysis and other technologies can facilitate determination of the nature of marker chromosomes.

Adult ; Chromosomes, Human, Y ; genetics ; Cytogenetics ; Humans ; In Situ Hybridization, Fluorescence ; Male ; Polymorphism, Single Nucleotide ; Sex Chromosome Aberrations ; Sex Chromosome Disorders ; genetics