Objective:To discuss the promotive effect of nerve growth factor(NGF),which is highly expressed in the adipose-derived stem cell(ADSC)-induced Schwann-like cells(SCLCs),on the growth of dorsal root ganglion(DRG)cell processes in the rats,and to clarify its mechanism.Methods:The ADSCs were extracted from the epididymal adipose tissue of the SD rats,and their multidirectional differentiation potential was identified through osteogenic,adipogenic,and chondrogenic induction.The ADSCs were induced to differentiate into the SCLCs,and the expression levels of glial fibrillary acidic protein(GFAP)and S100 calcium-binding protein β(S100β)protein in the ADSCs and SCLCs were detected by immunofluorescence staining and Western blotting methods.The DRG cells were isolated and cultured,and immunofluorescence staining was used to detect the βⅢ-tubulin expression in the DRG cells for identification.The SCLCs were co-cultured with the DRG cells(co-culture group),the single-culture DRG cells were regared as DRG group and toluidine blue staining was used to observe and measure the length of DRG cell processes under the optical microscope in co-culture group and DRG group.Small interfering RNA(siRNA)transfection was used to knock down NGF,and plasmid transfection was used to over-express NGF.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the NGF mRNA expression levels in the cells in various groups;enzyme-linked immunosorbent assay(ELISA)method was used to detect the NGF protein levels in the cell supernatants.The transfected SCLCs were co-cultured with DRG cells and divided into control group,siNC/vector group,NGF knockdown group(si-NGF group),and NGF over-expression group(oe-NGF group).The lengths of DRG cell processes in various groups were observed.Results:The primary ADSCs adhered within 24 h after seeding,with a small number of lipid droplets remaining.After 3 d of culture,the cells were mostly short spindle-shaped,fusiform,or polygonal,growing rapidly in a vortex pattern.After passaging,the cells exhibited a uniform morphology,appearing as long spindles arranged in a fish-school pattern.After 14 d of adipogenic induction,the cell morphology changed from spindle-shaped to flat-round,with translucent lipid droplets forming in the cytoplasm,which were stained red by Oil Red O.After 28 d of osteogenic induction,the cells appeared sand-like with blurred morphology,and calcified nodules were observed,which were stained red by Alizarin Red and deposited in the extracellular matrix.After 28 d of chondrogenic induction in a 3D culture system,millet-sized chondrogenic spheres formed.Frozen sections of the spheres were stained with Alcian Blue,and acidic mucopolysaccharides in the cartilage tissue were stained blue under the microscope.Under the fluorescence microscope,the third-passage purified ADSCs showed positive expression of CD29[fluorescein isothiocy anate(FITC)-labeled green fluorescence]and CD44(Cy3-labeled red fluorescence).The immunofluorescence staining results showed that GFAP was labeled with FITC(green fluorescence),and S100β was labeled with Cy3(red fluorescence).The Western blotting results showed that compared with ADSCs,the expression levels of S100β and GFAP proteins in the SCLCs were increased(P<0.05).The primary DRG cells began to adhere 6 h after conventional culture,and after 3 d,the cell bodies appeared round and bright,with two linear processes extending from them.Under fluorescence microscope,the cells positively expressed the neuron-specific marker βⅢ-tubulin,confirming that the isolated cells were DRG cells.Compared with the ADSCs,the NGF protein expression level in the SCLCs was increased(P<0.05).Compared with DRG group,the length of DRG cell processes in co-culture group was the highest when DRG cells and SCLCs were co-cultured at a 1∶2 ratio(P<0.05).The RT-qPCR results showed that compared with si-NC group,the expression levels of NGF mRNA in the cell supernatant in si-NGF-1,si-NGF-2,and si-NGF-3 groups were significantly decreased(P<0.05),with si-NGF-1 showing the highest knockdown efficiency,which was selected for subsequent experiments.The ELISA results showed that compared with si-NC group,the NGF levels in the cell supernatant of si-NGF-1,si-NGF-2,and si-NGF-3 groups were decreased(P<0.05).Compared with Vector group,the expression level of NGF mRNA and NGF protein level in the supernatant in oe-NGF group were increased(P<0.05).Compared with control group and siNC/vector group,the length of DRG cell processes in si-NGF group was decreased(P<0.05),while the length of DRG cell processes in oe-NGF group was increased(P<0.05).Conclusion:ADSCs can be directionally differentiated into SCLCs,and the differentiated cells highly express NGF.Knockdown or overexpression of NGF can affect the growth of DRG cell processes.

Objective:To observe the effects of adipose-derived stem cells(ADSC)combined with acellular scaffold(AS)on the ultrastructure of dorsal root ganglion and the protein and mRNA expression levels of ciliary neurotrophic factor(CNTF),Janus kinase 2(JAK2),phosphorylated JAK2(p-JAK2),signal transducer and activator of transcription 3(STAT3)and phosphorylated STAT3(p-STAT3)in the rats with sciatic nerve injury(SNI),and to clarify the protective effect of ADSC combined with AS on dorsal root ganglion in the SNI rats and its possible mechanism.Methods:The rat ADSCs were isolated and cultured and their multidirectional differentiation potential was detected.The AS of rats was prepared,and ADSCs were injected into the AS to construct tissue-engineered nerve.A total of 36 rats were randomly divided into control group,model group,AS group,and ADSC+AS group.The rats in control group were routinely fed,and the rats in other groups were used to establish the SNI models by resecting 10 mm of right sciatic nerve.The rats in model group received no further treatment,while the rats in AS group and ADSC+AS group were bridged with AS and the constructed tissue-engineered nerve at the two ends of the injured nerve,respectively.At 6 weeks after surgery,transmission electron microscope was used to observe the ultrastructure of dorsal root ganglion of the rats in various groups;immunofluorescence method was used to detect the protein expression levels of CNTF,p-JAK2,and p-STAT3 in dorsal root ganglion of the rats;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the mRNA expression levels of CNTF,JAK2,and STAT3 in dorsal root ganglion of the rats in various groups.Results:After 7 d of primary ADSC culture,a large number of large and long spindle-shaped cells were observed under the inverted microscope,arranged in clusters or whirlpools;red lipid droplets were observed with oil red O staining under microscope,and calcified nodules were observed with Alizarin red staining under microscope,indicating that the isolated and cultured cells had multidirectional differentiation ability.Compared with normal nerve tissue,the level of DNA in AS of rats was significantly decreased(P<0.05).Compared with control group,the nuclear membrane of dorsal root ganglion cells in model group was uneven and serrated,the number of organelles in the cytoplasm was decreased,mitochondria were swollen with broken or missing cristae and unclear structure;the CNTF protein and mRNA expression levels were significantly decreased(P<0.01),the p-JAK2 and p-STAT3 protein expression levels were significantly increased(P<0.01),and the JAK2 and STAT3 mRNA expression levels were significantly increased(P<0.01).Compared with model group,the serrated change of nuclear membrane of the dorsal root ganglion cells in AS group was significantly alleviated,the number of organelles in the cytoplasm was increased,and mitochondrial swelling was reduced;in ADSC+AS group,the nuclear membrane of dorsal root ganglion cells tended to be intact,the number of organelles was increased,and mitochondrial swelling and vacuolization were significantly reduced;the CNTF protein and mRNA expression levels in the dorsal root ganglion in AS group and ADSC+AS group were significantly increased(P<0.01),the p-JAK2 and p-STAT3 protein expression levels were significantly decreased(P<0.01),and the JAK2 and STAT3 mRNA expression levels were significantly decreased(P<0.01).Compared with AS group,the CNTF protein and mRNA expression levels in ADSC+AS group were significantly increased(P<0.05 or P<0.01),the p-JAK2 and p-STAT3 protein expression levels were significantly decreased(P<0.01),and the JAK2 and STAT3 mRNA expression levels were significantly decreased(P<0.01).Conclusion:The application of ADSC combined with AS can improve the ultrastructure of dorsal root ganglion in the SNI rats,and the mechanism may be related to the increased CNTF expression and decreased activation of the JAK2/STAT3 signaling pathway in the dorsal root ganglion by ADSC combined with AS application.

Using high-energy proton to image the region of interest can directly obtain the accurate estimation of the proton stopping power of the lesions,which is of great significance to reduce the range uncertainty in proton therapy.As a fundamental function of proton computed tomography(CT),radiographic imaging plays a crucial role in assisting clinical positioning.The study develops a compact proton CT detector based on an active array pixel CMOS chip in Monte-Carlo simulation toolkit Geant4,and evaluates the radiographic imaging capability of the system using 180 MeV protons.The angles of tracks are successfully reconstructed.CTP404,CTP528,and the CTP515 of specific materials are used for simulation,obtaining the spatial and density resolutions,and measuring the proton relative stopping power(RSP).The image signal-to-noise ratio is improved when using 2° proton scattering angle cut-off value.The spatial resolution is 3-4 lp/cm measured using CTP528 module.The density resolution is better than 0.05 g/cm3,and the RSP resolution is within 5%when CTP404 module is used.Through the imaging of CTP515 phantom of specific material,it is demonstrated that the system has potential for imaging common human tissues.

Transcription, Genetic

【Objective】 To explore the therapeutic effects of lactate dehydrogenase A (LDHA) inhibitor and targeted drugs on fumarate-hydratase-deficient renal cell carcinoma (FH-d RCC). 【Methods】 RNA-sequencing was used to detect the mRNA expression in FH-d RCC tissues, which was further validated with real-time fluorescence quantitative PCR and immunohistochemistry. Human-derived FH-d RCC cell line UOK262 and murine-derived FH-d RCC cell line FH1^-/-CL19 (CL19) were treated with LDHA inhibitor [(R)-GNE-140] and listed kidney cancer targeted drugs (Axitinib, Cabozantinib, Sunitinib, Sorafenib, Pazopanib, Everolimus) respectively, and then treated with LHDA inhibitor in combination with the targeted drugs to observe the alteration of cell proliferation. The combination index (CI) of different dose groups of the combination drugs were analyzed with CompuSyn software to determine the optimal combination regimen. 【Results】 LDHA inhibitor and targeted drugs, including Cabozantinib, Sorafenib and Sunitinib, had a significant inhibitory effect on the proliferation of FH-d RCC cells, and the combination of Cabozantinib and Sorafenib or Pazopanib had a significant anti-tumor effect. 【Conclusion】 LDHA inhibitor combined with targeted drugs can significantly inhibit the growth of FH-d RCC cells, indicating that LDHA may be a potential therapeutic target of FH-d RCC.

Objective:To estimate in-hospital mortality after knee replacement (KR) and to assess its trend and risk factors in China.Methods:We included patients undergoing KR in the Hospital Quality Monitoring System in China (2013-2019) to estimate in-hospital mortality after KR and assessed relation of patient's and hospital's characteristics (year of surgery, age, gender, marital status, primary indication, Charlson comorbidity index, geographic location, hospital type, hospital volume of KR, and surgery type) to in-hospital mortality using multivariable Poisson regression.Results:The annual amount of KR has increased from 20 307 in 2013 to 35 757 in 2019, and has maintained an upward trend for 7 years. The mean age of patients having KR increased from 64.9 years in 2013 to 66.6 years in 2019. Of the total 218 923 KRs, 63 deaths (0.29‰) occurred within 30 days before discharging. Older age was associated with higher in-hospital mortality ( P for trend <0.001). Male gender had higher incidence of in-hospital mortality compared with female [relative risk (RR), 2.5; 95% CI: 1.5, 4.1]. Single marital status was associated with higher, albeit non-statistically significant, in-hospital mortality than married patients (RR, 2.1; 95% CI: 0.9, 4.6). Higher Charlson comorbidity index was associated with increased risk of in-hospital mortality ( P for trend <0.001). Risk of in-hospital mortality decreased with more hospital-year knee replacement surgeries ( P for trend <0.001). In-hospital mortality varied by geographic regions, with the lowest mortality in East region (0.16‰), followed by South-West (0.31‰), South-Central (0.31‰), North region (0.33‰), North-West (0.54‰) and North-East (0.59‰). Conclusion:In-hospital mortality after KR in China was relatively low. Older age, male gender, higher Charlson comorbidity index and lower hospital-year knee replacement surgeries were risk factors for in-hospital mortality. The mortality varied greatly according to the geographic location of hospital.

Patellofemoral osteoarthritis (PFOA) is a subtype of knee osteoarthritis that has gained popularity in recent years due to its high prevalence and disease burden. The National Clinical Research Center for Geriatric Disorders (Xiangya Hospital) and the Joint Surgery Branch of the Chinese Orthopaedic Association, together with the editorial department of the Chinese Journal of Orthopaedics, convened a panel of orthopaedic experts to come up with guidelines. Subsequently, the Chinese clinical practice guideline for patellofemoral osteoarthritis (2020 edition) was officially released in September 2020. This is of utmost importance in standardizing the clinical diagnosis and treatment of PFOA in China. The present guideline focused on the diagnosis (symptoms, signs and imaging changes), non-surgical interventions (primary treatment and pharmacotherapy) and surgical interventions (repair and reconstruction) of PFOA. The present interpretation aims to address key emerging clinical issues in the diagnosis and treatment of PFOA in China.

【Objective】 To investigate the feelings of blood donors during blood donation and its influencing factors, thus providing references for high quality blood donation service for blood banks. 【Methods】 A self-developed questionnaire concerning voluntary blood donation was randomly issued by the research group and recruited investigators among blood donors in 6 street blood donation sites of Nanjing Red Cross Blood Center at weekends during December 1~23, 2018. The basic information of blood donors, the difference of feelings during blood donation and the influencing factors were analyzed by descriptive analysis, chi-square test and binary Logistic regression analysis, respectively. 【Results】 A total of 405 questionnaires were issued, and all 405 copies were valid, with the response rate at 100% (405/405). Among the valid questionnaires, 46.67% (189/405) were first-time blood donors, 27.65% (112/405) had donated for 2~3 times, and 25.68% (104/405) donated more than 4 times. 53.59%(97/181), 23.7%(43/181) and 22.65%(41/181) of the above three groups of blood donors worried about the needles, and 55.48%(81/146), 22.60%(33/146) and 21.92%(32/146) worried about the blood outflow, 52.69% (88/167), 28.14% (47/167), and 19.16% (32/167 )worried about the pain during blood collection (P<0.05). The anxiety of unqualified blood test was similar to that of poor service (P>0.05). There were differences in blood donation experience among the three groups (P<0.05), and gender (P<0.05) was its influencing factor showed by binary Logistic regression analysis. 【Conclusion】 The majority of blood donors, who have donated at the street sites of Nanjing, are worried during blood donation collection. Therefore, the blood centers should provide high-quality services and humanized nursing interventions as well as strengthen the citizens′ awareness of blood donation.

Objective:To estimate the burden of hip fractures and related factors among the Chinese middle-aged and elderly population.Methods:Based on the data of China Health and Retirement Longitudinal Study (CHARLS) in 2015, the burden of hip fractures among Chinese middle-aged and elderly population was evaluated as the years lived with disability (YLD) rate. The relationship between gender, age (45-49, 50-59, 60-69 and ≥70 years old), educational level (no education, elementary school, junior high school, high school and above), region (urban, rural), gross domestic production (GDP) per capita (low, medium and high) and geographic area (northern, eastern, south-central, northwest and southwest regions) and the YLD rate of hip fractures were analyzed, respectively.Results:Excluding items with missing basic information and hip fracture data, a total of 17,830 subjects (8,405 males and 9,425 females) were included in the study with average age 61.6±9.8 years (range 45 to 105 years). The average age in male was 61.9±9.6 years, and that in female was 61.2±10.1 years. The prevalence of hip fractures was 2.3% (410/17,830). The total YLD rate was [694/100,000 (95% Uncertainty Interval ( UI): 462/100,000, 989/100,000)]. The YLD rates were similar between male [693/100,000 (95% UI: 461/100,000, 988/100,000)] and female [696/100,000 (95% UI: 463/100,000, 992/100,000)] subgroups. The YLD rate of hip fractures was increasing with age, which reached at maximum of 1,155/100,000 (95% UI: 769/100,000, 1,646/100,000) for participants aged 70 years or above. The rate was gradually decreased with the upgrade of the educational level. The participants with high school education and above reached the lowest of 434/100,000 (95% UI: 289/100,000, 619/100,000). In addition, the YLD rate of hip fractures in rural areas [721/100,000 (95% UI: 480/100,000, 1,027/100,000)] was higher than that in urban areas [650/100,000 (95% UI: 433/100,000, 926/100,000)]. The YLD rate in areas with higher GDP per capita [545/100,000 (95% UI: 363/100,000, 777/100,000)] was lower than that in areas with lower GDP per capita [761/100,000 (95% UI: 506/100,000, 1,084/100,000)]. Moreover, the participants living in the Northwest region were with the highest YLD rate of hip fractures [1,056/100,000 (95% UI: 703/100,000, 1,506/100,000)], followed by the Southwest region [887/100,000 (95% UI: 590/ 100,000, 1,264/100,000)] and the Northeast region [317/100,000 (95% UI: 211/100,000, 452/100,000)]. Conclusion:Hip fractures exerted heavy burdens on the Chinese middle-aged and elderly population. The YLD rate of hip fractures varied according to geographical regions, greater age, rural areas, low educational levels and low GDP per capita. These related factors could affect the disease burden of hip fractures in China.

Objective To investigate the effect of plasma membrane-associated sialidase 3(NEU3) activity on the proliferation and apoptosis of osteosarcoma MG-63 cells in vitro. Methods MG-63 cells were cultured in vitro. Anti-NEU3 antibody(Ab)immunofluorescent staining was used to indicate the cellular locali-zation of NEU3 in MG-63 cells. The cells treated with 0 nmol/ L 2-deoxy-2,3-didehydro-N-acetyl neuraminic acid(DANA)or 0 μg/ ml anti-NEU3 Ab were used as blank control groups. The cells were treated with 10, 20,50 nmol/ L DANA,or 0. 5,1. 0,2. 0 μg/ ml anti-NEU3 Ab for 24 h or 48 h,respectively. The inhibition rates of the cell proliferation and cell apoptosis rates were measured with CCK-8 and flow cytometry. The expression levels of oncogene-related proteins,Ras protein and Bcl-2 protein,were detected by Western blotting. Results The immunofluorescence result showed that NEU3 was located in the cytoplasm of MG-63 cell. After treating with 0,10,20,50 nmol/ L DANA for 48 h,the inhibition rates of cell proliferation were 0, 15. 10％ ± 3. 23％ ,41. 46％ ± 2. 31％ ,64. 68％ ± 4. 12％ ,with significant statistical difference(F = 99. 90, P < 0. 001),and the following contrast between each two groups met the statistical significance(all P < 0. 05). After treating with 0,0. 5,1. 0,2. 0 μg/ ml anti-NEU3 Ab for 48 h,the inhibition rates of cell proliferation were 0,9. 34％ ± 1. 53％ ,19. 66％ ± 4. 18％ ,42. 50％ ± 5. 68％ ,and the difference was statistically signifi-cant(F = 25. 67,P < 0. 001),and the following contrast between each two groups met the statistical signifi-cance(P < 0. 05),except the difference between 0. 5 and 1. 0 μg/ ml groups(P > 0. 05). When the MG-63 cells were treated with 0,10,20,50 nmol/ L DANA for 24 h,the cell apoptosis rates were 4. 05％ ± 0. 07％ , 4. 15％ ± 0. 23％ ,12. 85％ ± 1. 48％ ,8. 29％ ± 0. 86％ ,respectively,and the difference was statistically sig-nificant(F = 23. 21,P < 0. 001). And the following contrast between each two groups met the statistical signi-ficance(P < 0. 05),except the differences between 0 nmol/ L and 10 nmol/ L,20 nmol/ L and 50 nmol/ L groups(P > 0. 05). When the MG-63 cells were treated with 0,0. 5,1. 0,2. 0 μg/ ml anti-NEU3 Ab for 24 h,the cell apoptosis rates were 4. 05％ ± 0. 07％ ,20. 13％ ± 2. 97％ ,20. 29％ ± 2. 82％ ,20. 58％ ± 0. 70％ ,with statistical significant difference(F = 15. 36,P = 0. 001). And the following contrast between each two groups showed that the differences between 0 μg/ ml and each treated group were statistically signifi-cant(P < 0. 05),while the differences between two treated groups were not statistically significant( P >0. 05). Western blotting results showed that the expression levels of Ras and Bcl-2 decreased with the increasing concentrations of DANA and anti-NEU3. Conclusion Inhibition of NEU3 enzyme activity can suppress the survival rate of MG63 cells and increase the cell apoptosis. The possible mechanism may be related to the declined expression of oncogene-related proteins Ras and Bcl-2,which suggests that NEU3 may be a possible target for treating osteosarcoma.