Objective To investigate the vascular endothelial growth factor (VEGF) expression level by chondrocytes isolated from patients with osteoarthritis (OA) in hip or femoral neck fracture (FNF) and explore the effect of synovial fluid from OA or FNF on secretion of VEGF. Methods The cartilage tissues were collected from 12 patients with OA in hip and 8 patients with FNF. Cartilage was stained with HIM and Safranin O/Fast Green (S/F) method. The damage of cartilage was evaluated using Mankin scores.Cathepsin B which was selected for cell dedifferentiation monitoring marker and VEGF level was detected in the supernatant fluid. The synovial fluid from OA, FNF and DMEM were respectively added to the culture medium to explore their effects on regulating VEGF. Results Cartilage the Mankin scores of OA group were higher than that of FNF group. Chondrocytes gradually lost their original spherical appearance, with Cathepsin B upregulated while VEGF downregulated. The OA synovial fluid can stimulate chongdrocytes to secrete more VEGF than the one from patients with FNF. However, chondrocytes gradually produced less VEGF after passaging. Conclusion Mankin scores had good correlation with chondrocytes' VEGF production in the early stage of primary culture. Chondrocytes showed quick dedifferentiation characteristics in vitro. OA synovial fluid showed abig ger capability in stimulating chondrocytes to express more VEGF, which might indicate that OA synovial fluid participated in the pathological process of OA.

Objective:To elucidate the effect of P2X4 signal axis on iron metabolism in the substantia nigra (SN) of male rats with Parkinson′s disease (PD) successfully induced by 6-hydroxydopamine (6-OHDA).Methods:A total of 120 male rats were randomly divided into control group, 6-OHDA group (PD group), P2X4-gene virus (P2X4-positive intervention, P2X4-PI) group, P2X4-gene unloaded virus (P2X4-negative control, P2X4-NC) group, P2X4-PI+6-OHDA group (inject P2X4 gene virus first, then 6-OHDA two weeks later) and P2X4-NC+6-OHDA group (inject no-load gene virus first, then two weeks later with 6-OHDA) using a completely random numbers method, with 20 rats in each group. Brain stereotactic instrument was used to inject the corresponding grouped drugs into the left SN of rats. A behavioral test was performed two weeks after the modeling was completed to select the qualified rat models, and the initiation and balance ability of each group of rats were further evaluated by a balance bar experiment. The brains of the qualified rat models were decapitated, and the brain tissue was taken away and preserved after related treatment. Immunofluorescence staining and Western blotting methods were used to detect the number of tyrosine hydroxylase (TH) positive dopaminergic neurons, and the expression levels of protein in P2X4 purinergic receptor (P2X4R), divalent metal-ion transporter-1 (DMT1) and ferroportin 1 (FPN1).Results:The results of immunofluorescence staining showed that the number of TH positive dopaminergic neurons in the left SN of the PD group (4 724.0±261.1, t=13.17, P<0.01) and the P2X4-NC+6-OHDA group (4 470.0±228.9, t=14.21, P<0.001) was significantly lower than that of the control group (7 942.0±461.6). The number of TH positive dopaminergic neurons of the P2X4-PI+6-OHDA group (2 493.0±371.6, t=8.092, P<0.01) was significantly lower than that of the P2X4-NC+6-OHDA group. The results of Western blotting suggested that compared with the control group (1.723±0.146, 1.369±0.107, 1.020±0.059), the expression of P2X4R, DMT1 was increased, whereas FPN1 was decreased in the left SN of the PD group (2.107±0.070, t=4.368, P<0.05; 1.733±0.117, t=4.245, P<0.05; 0.783±0.042, t=5.795, P<0.01) and the P2X4-NC+6-OHDA group (2.104±0.110, t=4.326; 1.737±0.073, t=4.291; 0.804±0.037, t=5.282; P<0.05). Compared with the P2X4-NC+6-OHDA group, the expression of P2X4R, DMT1 was increased and FPN1 was decreased in the left SN of the P2X4-PI+6-OHDA group (2.875±0.170, t=8.770; 2.845±0.180, t=12.92; 0.550±0.040, t=6.216; P<0.01). Conclusion:The overexpression of P2X4 gene can up-regulate the expression of DMT1 and down-regulate the expression of FPN1 in the SN, which leads to the deposition of iron in the SN of the midbrain, and then may cause damage to dopaminergic neurons, and finally has an effect on the occurrence and development of PD.

Objective To explore the prognostic factors of the pathological complete response of internal mammary lymph node (ipCR) after neoadjuvant chemotherapy and its effect on breast cancer prognosis. Methods We retrospectively analyzed the clinical data of 70 patients with primary breast cancer with internal mammary lymph node metastasis who received neoadjuvant chemotherapy. Patients were divided into the ipCR group and non-ipCR group based on their postoperative pathology. χ² test, Fisher, and Logistic regression were used for univariate and multivariate analysis. Meanwhile, the Kaplan-Meier curve and Cox regression were used for prognostic analysis. Results Of 70 patients, 31 obtained ipCR (44.3%). Univariate analysis showed that the expression levels of apCR, HR, and HER2 status were related to ipCR (P < 0.05). Multivariate analysis showed that age, apCR, and HER2 status were independent predictors of ipCR (P < 0.05). The average DFS of ipCR group was better than non-ipCR group (96.0 vs. 67.1 months, P < 0.05). The risk of recurrence and metastasis was 87% lower in the ipCR group than in the non-ipCR group (HR=0.13, 95%CI: 0.04-0.44, P < 0.01). ipCR, Ki67 expression level, and breast pCR (bpCR) were independent factors affecting patients' prognosis. Conclusion There is a correlation between clinico pathological factors and ipCR after neoadjuvant chemotherapy. ipCR can be used to predict the prognosis of patients with internal mammary lymph node metastasis.

Objective We report a case of application of third-generation sequencing(TGS)combined with preimplantation genetic testing(PGT)for successful prevention of hereditary spastic paraplegia(HSP)caused by SPAST gene mutations and assess the value of PGT-M and TGS in managing hereditary spastic paraplegia.Methods A family affected by HSP underwent whole exon sequencing(WES),and a c.1699G>T mutation in the SPAST gene was identified.The mutation site in the proband was confirmed through Sanger sequencing.A single nucleotide polymorphism(SNP)site flanking the SPAST gene mutation was selected as the genetic linkage marker,and a SNP haplotype carrying the mutated gene was constructed.Ovarian stimulation using an antagonist regimen was performed for oocyte retrieval,followed by intracytoplasmic sperm injection(ICSI)and embryo culture.Blastocyst trophectoderm cells were biopsied for preimplantation genetic testing for monogenic disorders(PGT-M)to allow the selection of disease-free embryos for transfer.Results In this cycle,a total of 20 oocytes were retrieved,among which 18 were successfully fertilized to result in 12 blastocysts eligible for biopsy.Genetic testing revealed that all the 12 blastocysts were successfully amplified and confirmed as euploidy.Among them,8 blastocysts did not carry paternal mutations,and a high-quality euploid embryo was selected for frozen embryo transfer(FET).Subsequent amniotic fluid testing during pregnancy confirmed the absence of paternal mutations in the fetus,resulting in the birth of a healthy baby girl.Conclusion For cases of genetic diseases with missing pedigree data,the application of third-generation sequencing and PGT-M technique can effectively block vertical transmission of SPAST gene mutation to the offspring,avoid pregnancy with an aneuploid embryo,and help families with autosomal dominant HSP obtain healthy offsprings.

Objective We report a case of application of third-generation sequencing(TGS)combined with preimplantation genetic testing(PGT)for successful prevention of hereditary spastic paraplegia(HSP)caused by SPAST gene mutations and assess the value of PGT-M and TGS in managing hereditary spastic paraplegia.Methods A family affected by HSP underwent whole exon sequencing(WES),and a c.1699G>T mutation in the SPAST gene was identified.The mutation site in the proband was confirmed through Sanger sequencing.A single nucleotide polymorphism(SNP)site flanking the SPAST gene mutation was selected as the genetic linkage marker,and a SNP haplotype carrying the mutated gene was constructed.Ovarian stimulation using an antagonist regimen was performed for oocyte retrieval,followed by intracytoplasmic sperm injection(ICSI)and embryo culture.Blastocyst trophectoderm cells were biopsied for preimplantation genetic testing for monogenic disorders(PGT-M)to allow the selection of disease-free embryos for transfer.Results In this cycle,a total of 20 oocytes were retrieved,among which 18 were successfully fertilized to result in 12 blastocysts eligible for biopsy.Genetic testing revealed that all the 12 blastocysts were successfully amplified and confirmed as euploidy.Among them,8 blastocysts did not carry paternal mutations,and a high-quality euploid embryo was selected for frozen embryo transfer(FET).Subsequent amniotic fluid testing during pregnancy confirmed the absence of paternal mutations in the fetus,resulting in the birth of a healthy baby girl.Conclusion For cases of genetic diseases with missing pedigree data,the application of third-generation sequencing and PGT-M technique can effectively block vertical transmission of SPAST gene mutation to the offspring,avoid pregnancy with an aneuploid embryo,and help families with autosomal dominant HSP obtain healthy offsprings.