Objective To explore the different methods of decoloration for immunohistochemical HE slides,and their influence on tissue antigen expression.Methods Antigen retrieval solution (EDTA or citric acid solution) was used in immunohistochemistry to decolor HE slides,and was compared with the method of hydrochloric acid solution decoloration in normal temperature.Results Antigen heat retrieval decolors completely.Compared with other methods,it has shorter decoloration time (1 ～ 2min),more than 90％ of completely decoloration rate,the difference was significant(x2 =27.6,23.9,all P ＜ 0.01).Meanwhile,the method dose not damage slides,and the immunohistoehemical results are exact,without background coloring and unusual coloring,antigen expression rate and intensity are high.Conclusion Antigen heat retrieval decoloration not only simplifies the coloration process,but also protects tissue antigen activity better.It is the preferable method of decoloration and protecting antigen resource,which possesses some practical value in the pathology diagnosis.

BACKGROUND: Graft selection and histological fate for anterior cruciate ligament reconstruction is a hot topic in the fields of reconstruction and repair of anterior cruciate ligament.OBJECTIVE: To review structure of anterior cruciate ligament and graft selection.METHODS: Articles were retrieved from Medline database with the key words of "Anterior cruciate ligament, implant,reconstruction" between January 1980 and January 2010. inclusion criteria: ① Reconstructive surgery of anterior cruciate ligament injury; ② graft selection of anterior cruciate ligament. Exclusion criteria: ① the old literatures; ② repetitive studies. A total of articles related to reconstruction of anterior cruciate ligament were retrieved, but 33 ones were included in the final analysis. The old, duplicated, and similar studies were excluded.RESULTS AND CONCLUSION: At present, the major therapy for anterior cruciate ligament injury includes arthroscopy and arthroscopy-assisted reconstruction. For clincal application, there are a lot of grafts, including autogenous grafts, allografts,heterologous allograft, biological materials, artificial materials and tissue engineering grafts. Autogenous semitendinosus tendon and gracilis tendon or autogenous bone-patellar tendon (middle 1/3)-bone (BPTB) are mainly used for anterior cruciate ligament reconstruction at home and abroad.

Objective To explore the effect of cryopreserved canine kidney cells (MDCK)single -layer on the isolation and proliferation of influenza virus B.Methods Revived P17 MDCK cells were passage for 2 -4 gener-ations,and subsequently preserved in 4℃ refrigerator for 3,6 and 9 days,respectively.Under same experimental con-ditions,the 4℃ refigerater preserved cells were co -incubated with influenza -like illness(ILI)throat swab speci-mens.Cytopathic effect (CPE)was observed,and the proliferation of virus was determined using real -time PCR and the hemagglutinin titers were determined by serological test.Results (1)CPE:The CPE of the MDCK cells pre-served in 4℃ refrigerator for 3 or 6 days had no significant differences compared with that in the control group,while the cell preserved in 4℃ refrigerator for 9 days showed CPE fastly and maintained for a short time.(2)Real -time PCR:the proliferation of influenza virus B in the MDCK cells preserved with 4℃refrigerator for 3 or 6 days (25.86 × 105 -30.25 ×106 ,26.31 ×105 -30.54 ×106 )had on difference compared with that of the control group (24.82 × 105 -29.86 ×106 ),with the proliferation rate of 105 to 106 times,while the proliferation cell with 4℃ cryopreserved for 9 days(19.72 ×104 -28.34 ×105 )the proliferation in cells preserved for 9 days was sharply decreased,with pro-liferation rate of 104 to 105 times.(3)The HA titer:The virus strains with hemagglutination titer above or equal to 116 (P >0.05)isolated with MDCK cells preserved in 4℃ refrigerator 3 or 6 days were not significantly different from that of the control group (10.92 ±0.79).And the cells with 4℃ cryopreserved for 9 days were significantly dicreased (P <0.01).Conclusion No significant effects on the isolation and proliferation of influenza virus B using MDCK cell preserved in 4℃ frigerator for near one week were observed in the present study.

Objective:To understand the genetic variation and the prevalence of H9N2 subtype avian influenza virus in Guizhou province, and to provide the scientific evidence for the prevention and control of avian influenza virus.Methods:The results of AIV detection in live poultry market(LPM) environment in Guizhou province from October 2018 to March 2019 were statistically analyzed, RNAs were extracted and sequenced from the HA genes of 13 samples of H9N2 positive screened by real-time PCR. Then the homology, the genetic evolution and the mutations of important amino acid were analyzed by bioinformation softwares. Results:The positive rate of AIV was 52.2% and the positive rate of H9N2 was 83.7% in LPM environment. The homology between nucleotides of the HA gene of 21 strains ranged from 91.6% to 100.0%, and the homology between amino acids of the HA gene ranged from 91.0% to 100.0%. All strains belonged to Y280 sublineage and G57 genotype. Key sites analysis showed that they had a common motif PSRSSRGLF and LSRSSRGLF at the cleavage site, which indicated that they were lentogenic and low pathogenic strains. Mutations H191N, E198T/A and Q234L at the receptor binding sites in the HA was found in 21 strains, while indicated the viruses had the potential to bind human-like receptor. The analysis results of glycosylation motifs showed that all 21 strains had 7 glycosylation sites, but had a site deletion at amino acid site 218 and an addition at 313.There was no significant mutation in the key site compared with the human infected strains. Conclusions:The detection rate of AIV in LPM environment in Guizhou province was high, and the pollution was very serious, and H9N2 subtype is the main subtype, All H9N2 subtype AIVs belonged to Y280 sublineage and G57 genotype, and they were low pathogenic avian influenza viruses in Guizhou province, but the genetic gap were widening and mutations of key amino acid site might enhance susceptibility and pathogenicity to human beings. Hence, It is necessary to strengthen the surveillance of molecular characteristic variation of H9N2 subtype AIV.

Objective To understand the epidemic characteristics and regularity of influenza B virus in Guizhou Province, and provide scientific evidence for the control and prevention of influenza.Methods Results of reverse transcription polymerase chain reaction(PT-PCR) of influenza B virus in Guizhou Province from April 1, 2013 to March 31, 2016 were statistically analyzed.Results A total of 1 904 samples were detected influenza B virus by RT-PCR, B/Yamagata (By) lineage and B/Victoria (Bv) lineage were 1 215 and 642 respectively.In April 2013-March 2014 and April 2014-March 2015, the predominant strains of influenza B were both By lineage, in April 2015-March 2016, the predominant strains of influenza B were Bv and By lineages, the epidemic peaks were in winter and spring;there's a higher positive percentage of influenza B in male, accounting for 56.83%;the highest detection rate of influenza B virus was found in population aged <15 years(70.80%),Bv and By lineages were the highest in the 0~ (42.37%) and 5~ age groups (35.56%) respectively;the main pathogen causing mixed infection was By+Bv (67.65%),mixed infection with influenza B virus accounted for 95.59%.Conclusion There are two lineages By and Bv epidemic in Guizhou Province, the epidemic peaks of influenza B are in winter and spring, male cases are higher than female, people under 15 years old are the high-risk group for influenza B, it is of great significance to strengthen the vaccination and surveillance of influenza in low age population.

Objective To understand the genetic variations of influenza B virus outbreaks in Guizhou province in 2016,and to compare the matching situation of outbreak epidemic strains with the vaccine strains recommended by WHO and representative strains in China.Methods The haemagglutinin HA1 gene of 8 strains isolated from two episodes of influenza B virus outbreaks in Tongren area was amplified and sequenced.The sequencing products were analyzed by bioinformatics software DNAStar. Results The two episodes of influenza outbreaks were both caused by influenza B Victoria lineage virus (BV).The homologies of the isolated strains were 99.8%—100.0% in nucleotide and 99.5 %—100.0%in amino acid.Mutation was only detected at 274 site in some strains.Compared with reference strain B/Victoria/2/87,the homologies were 91 .8%—92.0% and 91 .5 %—92.0%,respectively.Mutations developed at 17 amino acid sites,among which,I143V,V163I and V201I site were associated with the main antigenic determinant area B,C and D.Compared with previous vaccine strain B/Brisbane/60/2008, the homologies were 98.2%—98.3% and 98.5 %—99.0%,respectively,and mutations were detected at 3 sites.Mutations at I143V and N155D were detected in all 8 strains and at T247I in some strains.The mutation of I143V was associated with antigenic determinant area B.Compared with the representative strain B/Chongqing-Yuzhong/1384/2010,the homologies were 96.7%—96.8% and 97.0%—97.5 %, respectively.A total of 6 sites developed mutations,among which,5 sites were P84L,I143V,N155D, V172I and T223N mutations.The mutation of T247I was detected in some strains,and I143V was associated with area B.Compared with the epidemic strain in Guizhou in 2016,the homologies were 99.8%—100.0% and 99.5 %—100.0%,respectively.Mutation was only detected at site 247 in some strains and was not associated with the main antigenic determinant area.Conclusions The two episodes of influenza outbreaks in Guizhou are caused by the same BV lineage epidemic virus strain.Haemagglutinin gene of BV lineage virus is constantly changing.However,there is no new mutation emerged at important site.Compared with previous influenza vaccine strain B/Brisbane/60/2008 recommended by WHO,BV lineage virus is well matched and could provide a positive protection effect.

Objectives To investigate the prevalence of non-alcoholic fatty liver (NAFLD) disease and its correlation with chronic metabolic diseases in two primary school students in Shanghai. Methods One thousand ifve hundred and thirty-two 7-11 year-old students from two primary schools were enrolled in Septamber-October 2011. The anthropometric indices, blood pressure, screening for pseudoacanthosis nigricans and liver ultrasonography of all subjects were recorded. Results The overall prevalence of NAFLD, obesity, abdominal obesity, pseudoacanthosis nigricans, high systolic blood pressure and high diastolic blood pressure was 6.5%, 26.7%, 16.3%, 5.1%, 1.7%and 1.9%, respectively. The prevalence of NAFLD and abdomi-nal obesity in students of central urban area was signiifcantly higher than that in suburban area (P<0.01). The binary regression analysis revealed a signiifcant association between NAFLD in students with sex, age, obesity, abdominal obesity, pseudoacan-thosis nigricans and high systolic blood pressure (P<0.05). Conclusions NAFLD has close correlation with chronic metabolic disease in children. It is time to adopt prevention, detection and treatment in NAFLD children with symptoms of chronic meta-bolic diseases.

Objective To investigate the molecular characteristics of H5 subtype avian influenza viruses (AIV) in Weining, Guizhou Province. Methods Nine representative strains were randomly select-ed from H5 subtype AIV that were identified by real-time PCR in Weining, Guizhou Province from 2015 to 2017. Nucleic acid was extracted from each sample and hemagglutinin (HA) genes were amplified and then sequenced. Homology, genetic evolution and the sites related to pathogenicity, receptor binding regions as well as potential glycosylation of H5 AIV were analyzed by bioinformation software. Results Homology analysis revealed that there was 96. 1%-99. 9% and 95. 7%-100% similarity among the nine strains in nu-cleotide and amino acid of HA gene, respectively. These strains belonged to two branches, H5-1 and H5-2. The cleavage site motifs were PLREKRRKR↓GLF for five strains in H5-1 branch and PQRERRRKR↓GLF for four strains in H5-2 branch, which made them high pathogenic. QSG and QRG at the key receptor bind-ing sites were found in H5-1 and H5-2 branch strains, respectively. They were responsible for receptor bind-ing specificity of AIV. Mutations of 138Q, 139G and 53K were all detected in the nine strains. 129K, 189T, 140K and 282V mutations were discovered in the five strains of H5-1 branch, while 189N, 140M and 282I mutations were found in the four strains of H5-2 branch. Results of the glycosylation motif analysis showed that six sites were conservative, but there was an addition of 124NHT site in two strains of H5-2 branch isolated in 2017. Conclusion Two high pathogenic H5 subtypes of AIV could be epidemic in Wein-ing, Guizhou Province during 2015 to 2017. Although H5 subtype AIV did not possess specific receptor binding regions like human influenza viruses, they were in continuous variation with an increase in glycosyla-tion motifs, which might enhance their virulence and pathogenicity to human beings. Hence, surveillance and study on the molecular properties of H5 subtype AIV should be strengthened.

Objective To investigate the relationship between perineural invasion (PNI) and the clinicopathological features of cervical cancer,and to explore its influence on the prognosis of cervical cancer patients.Methods Retrospective analysis the clinical data of 76 patients with early cervical cancer who received radical hysterectomy + pelvic lymphadenectomy ± paraaortic lymphadenectomy from November 2015 to December 2019 in Xuanwu Hospital.The clinicopathological characteristics of the postoperative pathological results of cervical cancer patients were analyzed to explore the related factors of PNI and its influence on prognosis.Results Single factor analysis suggested that the incidence of PNI was related to tumor diameter,depth of stroma invasion,lymph-vascular space invasion,lymph node metastasis and clinical stage (P ＜ 0.05).Multivariate analysis suggested that the infiltration depth of stroma invasion was the risk factor of PNI (P ＜ 0.05).K-M analysis showed that there was no significant difference in disease-free survival and overall survival between PNI positive and PNI negative patients (P ＞ 0.05).Conclusions PNI is related to the depth of interstitial infiltration.PNI has no significant effect on the short-term recurrence rate and short-term survival rate of cervical cancer patients.

Objective: To investigate the molecular characteristics and tracing of the hemagglutinin (HA) gene, and to analyze the risk of human infection with influenza virus A (H7N9) in Guizhou Province, so that to provide evidence for the prevention and control of highly pathogenic avian influenza A (H7N9). Methods: Nucleic acids of 5 strains of H7N9 including 1 sample of the patient′s nasopharyngeal swab and 4 samples of the live poultry market (LPM) environment were extracted and HA genes were amplified and sequenced. Then the homology, genetic evolution and the pivotal sites related to receptor binding regions, pathogenicity and potential glycosylation of the avian influenza A (H7N9) viruses were analyzed by a series of bioinformatics softwares. Results: Homology analysis revealed that the homologies of nucleotide and amino-acid of the HA gene of H7N9 strains from the patient and LPM in Weining County, Guizhou Province were 99.8% and 99.6%, respectively, while those of 4 strains from LPM were both 100%. The homologies of nucleotide and amino-acid of the HA gene of H7N9 strains were the highest with the strain of A/Guangxi/5/2017 isolated from a Guangxi infected patient (99.7%-99.9% and 99.4%-99.8%, respectively), while those with the strain isolated from LPMs environment at the end of 2016 (A/Environment/Guangdong/C16283222/2016) were 99.0%-99.2% and 98.9%-99.2%, respectively. However, the homologies of nucleotide and amino-acid of the HA gene of H7N9 strains with A/Shanghai/2/2013 recommended by world health organization and the candidate vaccine strain A/Anhui/1/2013 were 96.8%-97.0% and 95.8%-96.2%, respectively. Phylogenetic analysis showed that the 5 strains had the nearest genetic distance to the strain A/Guangxi/5/2017. All the 5 strains cleavage site sequences of HA protein showed mutation of PEVPKRKRTAR↓GLF, and they were highly pathogenic avian influenza viruses mutant strains, which all had mutation of G186V at the receptor binding sites of HA gene, while no Q226L mutation was found. All 5 strains had new mutation of A363S, and new mutations of R56K and I297V were only found in the strain isolated from the patient. Among the five potential glycosylation motifs in the HA, only 421NWT and 493NNT had variation of the position post shift. Conclusions All the 5 H7N9 strains isolated in Weining County, Guizhou Province are highly pathogenic avian influenza mutative viruses. The current candidate vaccine may not provide a very good protection. The mutations of cleavage site of HA protein, G186V as well as other new mutation sites of HA may enhance the susceptibility and pathogenicity to human beings.