OBJECTIVE: To review the role and research progress of mechanotransduction signaling pathway in distraction osteogenesis, so as to provide theoretical basis and reference for clinical treatment. METHODS: The role and research progress of mechanotransduction signaling pathway in distraction osteogenesis were summarized by extensive review of relevant literature at home and abroad. RESULTS: The mechanotransduction signaling pathway plays a central role of "sensation-transformation-execution" in distraction osteogenesis, and activates a series of molecular mechanisms to promote the regeneration and remodeling of bone tissue by integrating external mechanical signals. Mechanical stimuli are converted into mechanotransduction signals through the perception of integrins, Piezo1 ion channels and bone cell networks. Activate downstream molecules are transduce through signal pathways such as Wnt/β-catenin, transforming growth factor β/bone morphogenetic protein-Smad, mitogen-activated protein kinase, protein kinase Hippo-Yes-associated protein/transcriptional coactivator with PDZ-binding motif, and phosphatidylinositol 3-kinase/ protein kinase B, so as to achieve the effects of promoting osteoblasts proliferation, accelerating endochondral ossification, regulating bone resorption and the like, thereby promoting the regeneration of new bone in the distraction area. The study of mechanotransduction signaling pathways in distraction osteogenesis is expected to optimize the mechanical parameters of distraction osteogenesis and provide targeted intervention strategies for accelerating new bone regeneration and mineralization in the distraction zone. However, the specific mechanism of mechanotransduction signaling pathway in distraction osteogenesis remains to be further elucidated, and artificial intelligence and multi-omics analysis may be the future development direction of mechanotransduction signaling pathway. CONCLUSION In distraction osteogenesis, mechanotransduction signal transduction is the core mechanism of bone regeneration in the distraction zone, which regulates cell behavior and tissue regeneration by converting mechanical stimulation into biochemical signals.
Mechanotransduction, Cellular/physiology* ; Osteogenesis, Distraction/methods* ; Humans ; Signal Transduction ; Bone Regeneration ; Animals ; Osteoblasts/metabolism* ; Osteogenesis ; Transforming Growth Factor beta/metabolism* ; Ion Channels/metabolism* ; Integrins/metabolism* ; beta Catenin/metabolism* ; Bone Morphogenetic Proteins/metabolism* ; Smad Proteins/metabolism*

Objective To establish a method of LC-MS/MS for determining cordycepin(Cor)and 3′-deoxyinosine(3′-Deo)concentration in rat plasma,and to study their pharmacokinetics in rats.Methods Protein was precipitated with methanol using 2-chloadenosine(2-Chl)as an internal standard.The chromatography was performed on Kinetex C18(3 mm×100 mm,2.6 μm,Phenomenex,USA)with gradient elution in aqueous(5 mmol·L-1 ammonium acetate)-methanol solution as mobile phase.ESI ion source was used for mass spectrometry,and positive ion multiple reaction monitoring(MRM)was used for scanning detection.The pharmacokinetics of Cor and 3′-Deo after oral administration of Cor(10 mg·kg-1)were studied in rats.Results Cor at 0.5-100 ng·mL-1 and 3′-Deo at 1-200 ng·mL-1 had good linearity,and the lower limits of quantification were 0.5 and 1 ng·mL-1,respectively.After oral administration of Cor in rats,the plasma concentration of Cor was low,which was mainly converted into the metabolite 3′-Deo.The Cmax of Cor and 3′-Deo were(5.4±3.4)and(142.0±50.0)ng·mL-1,and AUC0-360min min were(658.4±459.3)and(18 034.9±4 981.1)ng·min·mL-1,respectively.Conclusion The method is simple,sensi-tive,and accurate,which is suitable for determining Cor and 3′-Deo concentration in plasma and the pharmacokinetic study.

Objective: To investigate the degree and distribution of tissue-resident CD8+ T cell (CD103+CD8+T cells) infiltration in colorectal cancer (CRC) tissues, and to analyze its relationship to patients’clinicopathological features and prognosis. Methods: Tissue chips of 88 cases of colon cancer tissues (No.HColA180Su14) and 77 cases of rectal cancer tissues (No. HRec-Ade180Sur-03) were obtained from Shanghai Outdo Biotech Co.,Ltd. Immunofluorescence staining was performed to examine the infiltration pattern and degree of CD103+CD8+T cells in the collected CRC tissues and their para-cancerous tissues. Wilcoxon rank test was used to compare CD103+CD8+T cell infiltration degree in CRC tissues and the para-cancerous tissues. Chi-square test was used to analyze the relationship between CD103+CD8+T cell infiltration in CRC and patients’clinicopathological features. Kaplan-Meier survival analysis was conducted to explore the correlation between CD103+CD8+T cell infiltration and patients’prognosis. Cox model was applied to analyze the correlation between different clinical parameters and patients’prognosis. Results: CD103+CD8+T cell infiltration presented no signifi ·cant differences between CRC and para-cancer tissues (P>0.05). Patients with distant metastasis had significantly lower CD103+CD8+T cell infiltration rate than patients without distant metastasis (P<0.01). There was no significant correlation between the infiltration of CD103+CD8+T cells and other clinicopathological features (P>0.05). Kaplan-Meier survival analysis showed that the overall survival (OS) of patients with high CD103+CD8+T cell infiltration was significantly longer than that of the patients with low infiltration (54.42% vs 25.00%, P<0.05). Multivariate Cox model analysis indicated that pathological grade (P<0.01) and high CD103+CD8+T cell infiltration (P<0.05) were independent prognostic factors for CRC. Conclusion： ：CD103+CD8+T cell infiltration in CRC is associated with patients’prognosis, suggesting that CD103+CD8+T cell plays an important role in the initiation and development of CRC.

Objective: To investigate the degree of infiltration and distribution of tissue-resident CD8 + T cells (CD103 + CD8 + T cells) in gastric cancer tissues, and analyze the relationship between the degree of infiltration and clinicopathological features and prognosis. Methods: Tissue microarray and immunofluorescence staining were used to examine the CD8 + T cells and CD103 + CD8 + T cells infiltration in 90 cases of gastric cancer and their adjacent normal tissues. Wilcoxon rank test was used to compare the CD8 + T cells, CD103 + CD8 + T cells infiltration and CD103 + CD8 + T cells/CD8 + T cells ratio in gastric cancer and corresponding normal tissues. The chi-square test was used to analyze the relationship between CD8 + T cells, CD103 + CD8 + T cells infiltration and CD103 + CD8 + T cells/ CD8 + T cells ratio in gastric cancer tissues and clinicopathological features of the patients. Kaplan-Meier survival analysis was performed to explore the correlation between CD8 + T cells, CD103 + CD8 + T cells infiltration and CD103 + CD8 + T cells/ CD8 + T cells ratio and overall survival. Cox model was used to analyze the correlation between different clinical parameters and prognosis of the patients. Results: There was no significant difference for the infiltration of CD103 + CD8 + T cells between the gastric cancer tissues and adjacent normal tissues (P＞0.05). The infiltration rate of CD103 + CD8 + T cells in the cases in stage Ⅲ to Ⅳ (69.09%, 38/55) was significantly lower than that in the stage Ⅰ to Ⅱ cases (91.43%, 32/35), (χ 2 =6.175, P=0.013). There was no significant correlation between CD103 + CD8 + T cells infiltration and other clinicopathological features (P＞0.05). Kaplan-Meier survival analysis showed that the patients with high CD103 + CD8 + T cells infiltration showed significantly longer overall survival than the patients with low CD103 + CD8 + T cells infiltration (HR=2.187, 95%CI: 1.062-4.500, P=0.033 6). Multivariate Cox model analysis indicated that tumor diameter (HR=2.031, 95%CI: 1.163-3.546, P=0.013) and CD103 + CD8 + T cells infiltration (HR=0.516, 95%CI: 0.285-0.934, P=0.029) were independent prognostic factors for gastric cancer. Conclusion CD103 + CD8 + T cells in gastric cancer tissues should be associated with good prognosis, suggesting that they play an important role in the inhibition of gastric carcinogenesis and development, and can be used as an important factor for the prognosis evaluation of the patients with gastric cancer.

Objective: To investigate the expression of IFIT2 (interferon-induced protein with tetratricopeptide repeats 2) in human lung cancer tissue and analyze the relationship between the IFIT2 expression and clinicopathological features and prognosis. Methods: Tissue microarray and immunofluorescence staining were used to examine the IFIT2 expression in lung cancer tissue and their adjacent tissues. Wilcoxon rank test was used to compare the IFIT2 expression in lung cancer and corresponding adjacent tissues. The chi-square test was used to analyze the relationship between the IFIT2 expression in lung cancer tissues and clinicopathological features of the patients. Kaplan-Meier survival analysis was performed to analyze the correlation between IFIT2 expression and patients′ overall survival. Cox model was used to analyze the correlation between different clinical parameters and prognosis. Results: There was significant difference for the IFIT2 expression between the lung cancer tissues and adjacent tissues (P＜0.01). There was no significant correlation between IFIT2 expression and clinicopathological features of patients (P＞0.05). In lung adenocarcinoma and squamous cell carcinoma, Kaplan-Merier survival analysis showed that the overall survival (OS) of patients in IFIT2 low expression group was significantly shorter than that in IFIT2 high expression group (HR=2.392, 95%CI: 1.103-5.186, P=0.027; HR=2.907, 95%CI: 1.118-7.559, P=0.029, respectively). Multi-factor Cox model analysis indicated that distant metastasis (HR=8.033, 95% CI: 3.664-17.614, P=0.000) was independent prognostic factors for lung adenocarcinoma, lymph node metastasis (HR=3.390, 95% CI: 1.029-11.175, P=0.045) and IFIT2 low expression (HR=3.762,95%CI: 1.236-11.451, P=0.020) were independent prognostic factors for lung squamous cell carcinoma. Conclusion The down-regulated expression of IFIT2 in lung cancer tissues suggests that it may play an important role in initiation and development of lung cancer. It could be used as a valuable risk factor to predict the prognosis of lung cancer patients.

Objective To implement the finite discontinuity?volumetric modulated arc therapy ( FD?VMAT) in the Pinnacle planning system, and to investigate its clinical significance. Methods Eight patients with thoracic esophageal cancer in our hospital were enrolled as subjects. FD?VMAT was fulfilled in the Pinnacle planning system using a developed program. FD?VMAT, VMAT, and fixed?field intensity?modulated radiotherapy ( IMRT ) plans were designed for each patient. The conformity index ( CI ) and homogeneity index ( HI) of the planning target volume ( PTV) ,doses to organs at risk,passing rate for plan verification,number of monitor units,and treatment time were used to evaluate the plans. Comparison between different plans was made by paired t test. Results For the PTV,there was no significant difference in CI between FD?VMAT and VAMT ( P=0?186 );FD?VMAT had a significantly worse HI than VMAT ( P=0?001);however,both the CI and HI were significantly improved in FD?VMAT than in IMRT ( P=0?006, 0?002) . Compared with IMRT, FD?VMAT, retaining the advantage of VMAT, had pulmonary V20 and V30 significantly reduced by 19?79% and 20?32%,respectively (P=0?000,0?000).For the pulmonary low?dose regions (≤V5 ) ,FD?VMAT retained the advantage of IMRT and had lower doses than VMAT. Particularly, pulmonary V2 was significantly reduced by 16?79%(P=0?000).The mean lung dose was significantly lower in FD?VMAT than in VMAT or IMRT (P=0?001,0?000).There were no significant differences in D1cc to spinal cord PRV,heart V30,or passing rate for plan verification between the three therapies. The heart V40 and mean heart dose in FD?VMAT were similar to those in VMAT (P=0?175,0?468),but significantly lower than those in IMRT ( P=0?021,0?002) . FD?VMAT had a larger number of monitor units and longer treatment time than VMAT. Compared with IMRT, the number of monitor units and treatment time were reduced by 13?6% and 49?6% in FD?VMAT,respectively. Conclusions Compared with VMAT and IMRT, the application of the developed FD?VMAT in the treatment of thoracic esophageal cancer can further reduce the lung dose while keeping the PTV coverage,protection of the heart and spinal cord,and high efficacy. FD?VMAT is a new therapy available for thoracic esophageal cancer.

To enhance the specificity of anti-TNF-α single chain Fv antibody (TNF-scFv) to inflamed site, we constructed a bispecific antibody BsDb that targets TNF-α and ED-B-containing fibronectin (B-FN) by covalently linking TNF-scFv and the anti-ED-B scFv L19 at the gene level via a flexible peptide linker deriving from human serum albumin. BsDb was successfully secreted from Pichia pastoris as functional protein, identified by immunoblotting, and purified to homogeneity with affinity chromatography. BsDb retained the immunoreactivity of its original antibodies TNF-scFv and L19, and showed a marked gain in antigen-binding affinity and in TNF-α-neutralizing ability, when compared to TNF-scFv and L19 that were produced in Escherichia coli. In the adjuvant-induced arthritis (AIA) mice model, BsDb showed selective accumulation and retention in the inflamed paws but rapid clearance from blood, resulting in high arthritic paw to blood ratios. These data indicate that BsDb is endowed with high specificity to inflamed site and low toxicity to normal tissues and holds great potential for in vivo application for the targeted therapy of RA and other chronic inflammatory diseases.
Animals ; Antibodies, Bispecific ; biosynthesis ; immunology ; Antibodies, Neutralizing ; biosynthesis ; immunology ; Escherichia coli ; Fibronectins ; chemistry ; immunology ; Humans ; Mice ; Single-Chain Antibodies ; biosynthesis ; immunology ; Tumor Necrosis Factor-alpha ; immunology

Objective To explore the resistance of neural stem cells (NSCs) in β-amyloid (Aβ) toxicity after being transfected with brain-derived neurotrophic factor (BDNF) and its mechenism.Methods NSCs in the C57 mice were transfected with BDNF gene by using lipofectamine technique; the BDNF gene expression in transfected NSCs and their differentiated cells were detected by immune-fluorescent staining and ELISA.Rat neurons were cultured in vitro and divided into 3 groups:neurons group,neurons co-cultured with NSCs group,neurons co-cultured with BDNF transfected NSCs group; two-parallel group design was adopted.Three d after the group culturing,cells from each parallel group were added Aβ40 protein.Neuron survival of each group was recorded 24 and 48 h after adding Aβ40 by ELISA.The Aβ,tau,and phosphorylating tau (ptau) levels were detected by Western blotting.Results BDNF-transfected NSCs could continuously express BDNF gene products during the first 1.5 weeks.Both transfected NSCs and their progenies expressed BDNF gene products.Using neurons group as reference,the mean survival rates of neurons plus Aβ40 group,neurons co-cultured with NSCs plus Aβ40 group,neurons co-cultured with BDNFtransfected NSCs plus Aβ40 group were 51.86％±0.03％,73.68％±0.04％ and 85.34％±0.01％ at 24 h after adding Aβ40,and 38.76 ％±0.01％,59.65 ％±0.04％,75.61％± 0.07％ at 48 h after adding Aβ40; the survival rate in the neurons plus Aβ40 group and neurons co-cultured with NSCs plus Aβ40 group was significantly lower than the other groups (P＜0.05).High level of Aβ protein was noted in the neurons plus Aβ40 group,neurons co-cultured with NSCs plus Aβ40 group and neurons co-cultured with BDNF transfected NSCs plus Aβ40 group,without significant differences between each two groups (P＞0.05).The tau protein level showed no significant differences between each two groups (P＞0.05),while the p-tau protein level was significantly different (that in the neurons co-cultured with NSCs plus Aβ40 group and neurons co-cultured with BDNF transfected NSCs plus Aβ40 group was significantly higher than that in the other groups,P＜0.05).Conclusion BDNF-transfected NSCs express BDNF gene products during the early time,which alleviate the toxicity of Aβ to NSCs and reduce the p-tau protein level,leading to increased survival rate of neurons.

OBJECTIVETo construct a prokaryotic expression vector of human tau multiepitope peptide for examining the immunogenicity of a TauP1/P2 DNA vaccine in mice using the expressed product.

METHODSThe coding sequence of Tau multiepitope peptide gene was amplified from the plasmid pVAX1-Tau by PCR and inserted into the prokaryotic expression vector pGEX-4T-2 to construct the recombinant plasmid pGEX-4T-2-TauP1/P2. The positive recombinants were transformed into E.coli BL21 cells, and the expression of fusion protein GST-TauP1/P2 was induced by IPTG and identified by SDS-PAGE. Mice was immunized with TauP1/P2 DNA vaccine and the production of the specific antibodies was detected by Dot-blot analysis using the purified fusion protein.

RESULTSA gene fragment 300 bp in length was amplified. Enzyme digestion and DNA sequencing verified correct construction of the prokaryotic expression plasmid pGEX-4T-2-TauP1/P2. The expression of target fusion protein GST-TauP1/P2 was detected by SDS-PAGE. Specific antibodies against TauP1/P2 were detected in the serum of mice immunized with the DNA vaccine using GST-TauP1/P2 fusion protein.

CONCLUSIONThe constructed prokaryotic expression plasmid of human Tau multiepitope peptide is capable of expressing the target fusion protein, which specifically recognizes the specific antibodies against TauP1/P2 in mice immunized with TauP1/P2 DNA vaccine.

Animals ; Epitopes ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Mice ; Mice, Inbred C57BL ; Peptides ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, DNA ; biosynthesis ; immunology ; tau Proteins ; biosynthesis ; genetics ; immunology

ObjectiveTo evaluate whether oral administration of Chinese tradiational medicine,Qing-Xue granula,can prevent mouse lung injury caused by thoracic radiation.Methods128 BalB/C mice were divided into 4 groups:control (C) group; radiation (R) group; radiation plus high dose Qing-Xue granula (H) group and radiation plus median dose Qing-Xue granula ( M ) group.The H and M groups were fed 0.64 g and 0.32 g of Qing-Xue granula dissolved in 0.5 nl anline once daily for two months,which were 4 and 2 times of human dosage,respectively.Whole thorax radiation of 12 Gy was delivered with a single ventral-dorsal field with 6 MV X-ray.Group C and group R received 21 days of 0.5 ml saline feeding.Mice were sacrificed at 1,2,4 or 6 months after radiation. Macrophage cell count of lung lavage fluid and hydroxyproline content of left lung were assayed,and the lung fibrosis was scorred according to the Ashcroft's criteria.The plasma interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) concentration were assayed with ELISA method.The One-way ANOVA was used to test the significance of any differences between groups at each time point. Results The macrophage cell number of lung lavage fluid was significantly lower in the 1st month in group M than in group R (2∶4,q =3.92,P ＜ 0.05 ),but had no significant difference between group M and C ( 1 ∶ 4,q =2.13,P＞0.05 ).The hydroxyproline content of group H was significantly lower than group R in the 1st and 6th months (q =3.62,3.54,all P ＜ 0.05 ),but still higher than group C ( q =4.09,3.72,all P ＜ 0.05 ).The fibrosis score of group H was significantly lower than group R in the 2nd,4th and 6th months (q=3.38 -4.16,all P＜0.05).The IL-6 concentration of group H was significantly lower than group R in the 1st month ( q=3.53,P＜0.05 ),but not significantly higher than group C (q =1.41,P＞0.05).The VEGF concentration was significantly higher in group R than group C since the 2nd month ( q =3.12 - 3.78,P ＜ 0.05 ).The VEGF concentration was significantly higher in group H and M than group R in the 2nd and 6th months ( q =3.08 - 3.92,all P ＜ 0.0 5 ).Conclusions Oral Chinese traditional medicine,Qing-Xue granula,could prevent radiation induced lung fibrosis in mice,especially at high dosage.The degree of elevation of VEGF in plasma was not parallel with that of lung fibrosis.