1.NK cell-specific knockout of UTX modulates pulmonary metastasis of melanoma in a sex-dependent manner
Pei HUANG ; Hongchen WANG ; He HUANG ; Jiaxin XIE ; Yu WU ; Simin ZHOU ; Xinyi LIAO ; Xiao GUAN
Journal of Army Medical University 2025;47(8):807-815
Objective To explore the role of X chromosome encoded epigenetic regulator UTX in NK cell-mediated anti-tumor activity.Methods Male Ncr1-iCre mice were crossed with female UTXfl/fl mice to generate F1 Ncr1-iCre+UTXfl/-male mice,which were further crossed with female UTXfl/fl mice to obtain male Ncr1-iCre-UTX fl/-control mice(M-Con)and NK-specific deletion of UTX male mice Ncr1-iCre+UTXfl/-(M-KO),as well as female Ncr1-iCre-UTXfl/fl control mice(F-Con)and UTX-deficient female mice Ncr1-iCre+UTXfl/fl(F-KO).UTX-deficient mice were injected with melanoma cell line B16F10 via tail vein to observe pulmonary metastatic tumor nodules.Moreover,flow cytometry was applied to detect the proportion and quantity of pulmonary NK cells(CD3-CD19-NK1.1+),maturation makers KLRG1 and CD11b,activation receptors NKG2D and CD69,and effector molecules,including perforin,granzyme B,CD107a,and IFN-γ.Then pulmonary NK cells were sorted and co-cultured with B16F10 cells,and the apoptosis of the melanoma cells was measured with flow cytometry.Results Compared with the M-Con mice,the M-KO mice presented less number of pulmonary tumor nodules(P<0.05),increased proportion and quantity of NK cells in the tumor microenvironment(P<0.01),though no obvious changes in the ratio of NK maturation makers KLRG1 to CD11b,enhanced expression level of cytotoxic molecule perforin(P<0.01),but no changes in the expression of effector molecule granzyme B,degranulation marker CD107a and cytokine IFN-γ in NK cells.Co-culture of NK cells and B16F10 cells promoted the apoptosis of tumor cells(P<0.05).Compared with the F-Con mice,the F-KO mice had no statistical difference in the number of pulmonary tumor nodules,but larger proportion and number of NK cells(P<0.05),decreased ratio of KLRG1 to CD11b(P<0.01),elevated level of perforin but decreased levels of granzyme B,CD107a and IFN-γ in NK cells(P<0.01).The co-culture of NK cells and B16F10 cells reduced the apoptosis of tumor cells in F-KO female mice(P<0.05).Conclusion NK-specific deletion of UTX regulates pulmonary metastasis of melanoma in a sex-dependent manner.
2.Study on mechanism of electroacupuncture inhibition of NF-κB/NLRP3 signaling pathway in reducing pain in rats with knee osteoarthritis
Fang FENG ; Aimin ZHANG ; Hongchen HE
International Journal of Traditional Chinese Medicine 2025;47(11):1553-1560
Objective:To explore the relief effect and mechanism of electroacupuncture in rats with knee osteoarthritis (KOA) based on the NF-κB/NLRP3 signaling pathway.Methods:SD rats were divided into a blank control group, a model group, an electroacupuncture group, and an electroacupuncture+PMA group using a random number table method, with 10 rats in each group. Except for the blank control group, all other groups were used to prepare KOA models. After successful modeling, rats in the electroacupuncture group and electroacupuncture+PMA group were subjected to electroacupuncture stimulation on both sides of the "Yanglingquan"(GB34), "Neixiyan"(EX LE4), and "Waixiyan"(EX-LE5); the electroacupuncture+PMA group received intraperitoneal injection of 5 mg/kg PMA once a day for 11 consecutive days of intervention; the blank control group and model group did not receive any intervention. Thermal foot contraction latency (TWL) and mechanical foot contraction threshold (MWT) were detected. HE staining was used to observe the inflammation and damage of synovial tissue and knee articular cartilage tissue. ELISA was used to detect the levels of calcitonin gene-related peptide (CGRP), pain mediator substance P (SP) and IL-6 in serum. The expressions of CGRP, iNOS proteins in dorsal root ganglion were detected by immunofluorescence assay. The expressions of CGRP, iNOS, p-p65/p65, NLRP3, Caspase-1, ASC proteins in dorsal root ganglia were detected by Western blot.Results:Compared with the model group, the TWL and MWT of the rats in the electroacupuncture group (3, 6, 9 and 11 days) significantly increased ( P<0.05), the pathological damage of synovial tissue and knee articular cartilage tissue were reduced, serum CGRP, SP, IL-6 levels significantly decreased ( P<0.05), and the expressions of CGRP, iNOS, p-p65/p65, NLRP3, Caspase-1, ASC proteins in dorsal root ganglion significantly decreased ( P<0.05); PMA significantly inhibited the effect of electrolysis on KOA rats ( P<0.05). Conclusion:Electroacupuncture can alleviate pain symptoms in KOA rats, and the mechanism may be related to the inhibition of the activation of NF-κB/NLRP3 signaling pathway.
3.Development and external validation of a quantitative diagnostic model for malignant gastric lesions in clinical opportunistic screening: A multicenter real-world study
Hongchen ZHENG ; Zhen LIU ; Yun CHEN ; Ping JI ; Zhengyu FANG ; Yujie HE ; Chuanhai GUO ; Ping XIAO ; Chengwen WANG ; Weihua YIN ; Fenglei LI ; Xiujian CHEN ; Mengfei LIU ; Yaqi PAN ; Fangfang LIU ; Ying LIU ; Zhonghu HE ; Yang KE
Chinese Medical Journal 2024;137(19):2343-2350
Background::Clinical opportunistic screening is a cost-effective cancer screening modality. This study aimed to establish an easy-to-use diagnostic model serving as a risk stratification tool for identification of individuals with malignant gastric lesions for opportunistic screening.Methods::We developed a questionnaire-based diagnostic model using a joint dataset including two clinical cohorts from northern and southern China. The cohorts consisted of 17,360 outpatients who had undergone upper gastrointestinal endoscopic examination in endoscopic clinics. The final model was derived based on unconditional logistic regression, and predictors were selected according to the Akaike information criterion. External validation was carried out with 32,614 participants from a community-based randomized controlled trial.Results::This questionnaire-based diagnostic model for malignant gastric lesions had eight predictors, including advanced age, male gender, family history of gastric cancer, low body mass index, unexplained weight loss, consumption of leftover food, consumption of preserved food, and epigastric pain. This model showed high discriminative power in the development set with an area under the receiver operating characteristic curve (AUC) of 0.791 (95% confidence interval [CI]: 0.750–0.831). External validation of the model in the general population generated an AUC of 0.696 (95% CI: 0.570–0.822). This model showed an ideal ability for enriching prevalent malignant gastric lesions when applied to various scenarios.Conclusion::This easy-to-use questionnaire-based model for diagnosis of prevalent malignant gastric lesions may serve as an effective prescreening tool in clinical opportunistic screening for gastric cancer.
4.Research on clinical application of urine sediment score in the diagnosis of acute kidney injury
Hui ZHANG ; Wei XU ; Linlin QU ; Chunhe ZHAO ; Hongli SHAN ; Qin ZHANG ; Hongchen GAO ; Wenrui SUN ; Lina ZHU ; Yue ZHANG ; Xin YAN ; Xiaoquan YANG ; Wanning WANG ; Dong ZHANG ; Yao FU ; Xu ZHAO ; Liang HE
Chinese Journal of Laboratory Medicine 2024;47(5):548-553
Objective:To evaluate the clinical application of urine sediment score (USS) in early diagnosis, etiological differentiation, staging and prognosis of acute kidney injury (AKI), and to investigate the diagnostic efficacy of independent USS and its combination with blood urea nitrogen(Bun) serum creatinine(sCr) and uric acid(UA) in AKI.Methods:From August 23 to September 28, 2023, 9 020 morning urine samples of hospitalized patients in the First Hospital of Jilin University were detected by Sysmex UF5000.A total of 3 226 ssamples with small and round cell (SRC) > 1/μl and/or CAST>1/μl were screened for microscopic examination, and 404 cases with positive renal tubular epithelial cells and/or cast were enrolled in this study. There were 218 males and 186 females, aged 59.5 (49.0, 71.0) years. The 404 cases were divided into the USS AKI group (345 cases) and the USS non-AKI group (59 cases) according to the USS results based on the microscopic findings. According to Kidney Disease: Improving Global Outcomes (KDIGO) criteria, they were divided into KDIGO criteria AKI group (63 cases) and KDIGO criteria non-AKI group (341 cases), and the AKI group was divided into renal AKI group (33 cases) and non-renal AKI group (30 cases). According to the clinical diagnosis recorded in the medical records, they were divided into clinically diagnosed AKI group (29 cases) and clinically diagnosed non-AKI group (375 cases).The χ 2 test or Fisher exact test was used to compare USS in different AKI causes and stages. Logistic regression was used to calculate the odds ratio of renal AKI and stage 3 AKI. The area under the receiver operating characteristic curve was used to evaluate the sensitivity and specificity of USS, sCr, UA and Bun alone and in combination in the diagnosis of AKI, and the best cut-off value, sensitivity and specificity in the diagnosis of AKI were calculated. P < 0.05 was considered statistically significant. Results:The USS was used to identify the etiology of KDIGO standard AKI group,and there were significant differences in USS between renal AKI group and non-renal AKI group (χ 2=11.070, P<0.001). Compared to USS=1, the odds ratio of renal AKI was 8.125 when USS≥2 (95% CI 2.208—29.901). There was a statistically significant difference in the comparison of USS between groups in each stage of the AKI staging study based on USS (χ 2=15.724, P<0.05). Compared to USS=1, the odds ratio of stage 3 AKI was 9.714 when USS≥2 (95% CI 1.145-82.390). The AUC of independent USS in the diagnosis of AKI was 0.687 (95% CI 0.618-0.757, P<0.001), the specificity was 65.7% and the sensitivity was 61.9%. The AUC of USS combined with Bun, sCr, UA in the diagnosis of AKI was 0.794 (95% CI 0.608-0.980, P<0.05), the specificity was 82.4%, and the sensitivity was 88.9%. Conclusions:There wasan increased likelihood of renal AKI or stage 3 AKI while USS≥2,and whose combination with Bun, sCr and UA will improve the diagnostic efficiency of AKI.
5.Expert consensus on the biobank development of oral genetic diseases and rare diseases and storage codes of related biological samples from craniofacial and oral region
Wenyan RUAN ; Yanli ZHANG ; Shuguo ZHENG ; Yao SUN ; Zhipeng FAN ; Yaling SONG ; Hongchen SUN ; Wenmei WANG ; Jiewen DAI ; Zhenjin ZHAO ; Tingting ZHANG ; Dong CHEN ; Yongchu PAN ; Yuegui JIANG ; Xudong WANG ; Liwei ZHENG ; Qinglin ZHU ; Miao HE ; Baoshan XU ; Zhonglin JIA ; Dong HAN ; Xiaohong DUAN
Chinese Journal of Stomatology 2023;58(8):749-758
The biological samples of oral genetic diseases and rare diseases are extremely precious. Collecting and preserving these biological samples are helpful to elucidate the mechanisms and improve the level of diagnose and treatment of oral genetic diseases and rare diseases. The standardized construction of biobanks for oral genetic diseases and rare diseases is important for achieving these goals. At present, there is very little information on the construction of these biobanks, and the standards or suggestions for the classification and coding of biological samples from oral and maxillofacial sources, and this is not conducive to the standardization and information construction of biobanks for special oral diseases. This consensus summarizes the background, necessity, principles, and key points of constructing the biobank for oral genetic diseases and rare diseases. On the base of the group standard "Classification and Coding for Human Biomaterial" (GB/T 39768-2021) issued by the National Technical Committee for Standardization of Biological Samples, we suggest 76 new coding numbers for different of biological samples from oral and maxillofacial sources. We hope the consensus may promote the standardization, and smartization on the biobank construction as well as the overall research level of oral genetic diseases and rare diseases in China.
6.Effect of pulsed electromagnetic fields on mesenchymal stem cell-derived exosomes in inhibiting chondrocyte apoptosis.
Yang XU ; Qian WANG ; Xiangxiu WANG ; Xiaona XIANG ; Jialei PENG ; Jiangyin ZHANG ; Hongchen HE
Journal of Biomedical Engineering 2023;40(1):95-102
The study aims to explore the effect of mesenchymal stem cells-derived exosomes (MSCs-Exo) on staurosporine (STS)-induced chondrocyte apoptosis before and after exposure to pulsed electromagnetic field (PEMF) at different frequencies. The AMSCs were extracted from the epididymal fat of healthy rats before and after exposure to the PEMF at 1 mT amplitude and a frequency of 15, 45, and 75 Hz, respectively, in an incubator. MSCs-Exo was extracted and identified. Exosomes were labeled with DiO fluorescent dye, and then co-cultured with STS-induced chondrocytes for 24 h. Cellular uptake of MSC-Exo, apoptosis, and the protein and mRNA expression of aggrecan, caspase-3 and collagenⅡA in chondrocytes were observed. The study demonstrated that the exposure of 75 Hz PEMF was superior to 15 and 45 Hz PEMF in enhancing the effect of exosomes in alleviating chondrocyte apoptosis and promoting cell matrix synthesis. This study lays a foundation for the regulatory mechanism of PEMF stimulation on MSCs-Exo in inhibiting chondrocyte apoptosis, and opens up a new direction for the prevention and treatment of osteoarthritis.
Animals
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Rats
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Apoptosis
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Chondrocytes
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Electromagnetic Fields
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Exosomes/physiology*
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Mesenchymal Stem Cells/metabolism*
7.Application of platelet-rich plasma-based regenerative rehabilitation technology in the management of sports injury
Chinese Journal of Trauma 2023;39(9):780-785
The most common sports injury is trauma to the skeletal muscles, with injuries to ligaments, muscles, tendons, joint capsules or cartilage and minor injuries to other chronic soft tissues, accounting for 97% of all cases. These injuries directly affect athletes′ performance, shorten their athletic career, and also impede the daily life and work of the general public. Surgical and non-surgical interventions often fail to meet the demands for quick recovery, and patients with sports injury may experience joint stiffness, limited range of motion, and pain-related functional impairments. Platelet-rich plasma (PRP) has a role in the promotion of tissne regeneration, regulation of immune response and suppression of inflammation. PRP-based regenerative rehabilitation technology offers new possibilities for rapid recovery of patients with sports injury. However, there have been few reports on the combined application of PRP and principles/methods of rehabilitation medicine. To this end, the authors summarized the history of regenerative rehabilitation medicine, explored the feasibility of PRP-based regenerative rehabilitation technology in the treatment of sports injuries, and discussed the application prospects of PRP-based regenerative rehabilitation technology regarding the joint, muscle, tendon, and ligament injuries.
8.Early clinical efficacy of ultrasound-guided platelet-rich plasma technology in the treatment of lumbodorsal myofascial pain syndrome after sports injury
Shaolong AI ; Qian WANG ; Kaiwen LI ; Xingzhen LIN ; Na LI ; Hongying JIANG ; Hongchen HE
Chinese Journal of Trauma 2023;39(9):786-792
Objective:To explore the early clinical efficacy of ultrasound visualized platelet-rich plasma (PRP) in the treatment of lower back myofascial pain syndrome (MPS) after sports injury.Methods:A prospective cohort study was conducted to analyze the clinical data of 32 patients with lower back MPS after sports injury, who were admitted to West China Hospital of Sichuan University from January 2023 to March 2023. Ultrasound-guided PRP injection into the erector spinalis or quadratus psoas muscles was used for treatment. Before treatment, at 24 hours, 2 weeks, and 4 weeks after treatment, pain and function were evaluated using visual analogue scale (VAS), McGill pain questionnaire (McGill), Roland Morris dysfunction questionnaire (RMDQ), and Oswestry dysfunction index (ODI). Before treatment and 4 weeks after treatment, the quality of life was evaluated using the short-form 36 item health survey questionnaire (SF-36). The adverse reactions were observed during treatment and follow-up.Results:A total of 32 patients with lower back MPS after sports injury were enrolled, including 10 males and 22 females; aged 12-68 years [(47.3±16.3)years]. All the patients were followed up for 4 weeks. Before and at 24 hours, 2 weeks, and 4 weeks after treatment, the VAS was 5.0(4.0, 6.0)points, 3.5(3.0, 4.8)points, 2.0(2.0, 3.0)points, and 2.0(1.3, 3.0)points, respectively; the McGill score was 9.0(7.0, 11.0)points, 7.0(5.0, 9.0)points, 4.0(3.0, 5.0)points, and 3.0(3.0, 5.0)points, respectively; the RMDQ score was 8.0(5.3, 10.8)points, 5.5(3.0, 8.0)points, 4.0(3.0, 5.8)points, and 3.0(2.0, 4.8)points, respectively; the ODI was 22.0(14.5, 30.0), 20.0(14.5, 25.5), 9.0(6.0, 16.0), and 8.0(4.5, 14.0), respectively. Compared with the values before treatment, the VAS, McGill score, and RMDQ score were significantly decreased at 24 hours, 2 weeks, and 4 weeks after treatment (all P<0.05); the ODI had no significant difference at 24 hours after treatment ( P>0.05), but it was significantly decreased at 2 and 4 weeks after treatment (all P<0.05). Compared with the values at 24 hours after treatment, the VAS, McGill score, RMDQ score and ODI further decreased at 2 weeks after treatment (all P<0.05). Compared with the values at 2 weeks after treatment, there was no significant difference in the VAS, McGill score, RMDQ score, or ODI at 4 weeks after treatment (all P>0.05). In the SF-36, the scores of physiological function [77.5(60.0, 93.8)points], physiological role [50.0(0.0, 100.0)points], body pain [64.0(44.5, 74.0)points], vitality [75.0(65.0, 78.8)points], social function [87.5(75.0, 100.0)points], emotional role [66.7(33.3, 100.0)points] and mental health [72.0(68.0, 83.0)points] before treatment were increased to 90.0(80.0, 98.8)points, 100.0(56.3, 100.0)points, 84.0(74.0, 84.0)points, 75.0(70.0, 80.0)points, 100.0(87.5, 112.5)points, 100.0(66.7, 100.0)points, and 76.0(68.0, 84.0)points after 4 weeks of treatment, respectively ( P<0.05 or 0.01). However, there was no significant difference in the general health status or health changes before and after treatment (all P>0.05). During treatment and follow-up, no adverse reactions such as redness, swelling, pain, or subcutaneous bleeding were observed. Conclusion:Ultrasound-guided PRP treatment can improve the early pain, lumbar mobility and quality of life of patients with lower back MPS after sports injury, with no presence of adverse reactions.
9.Negative pressure wound therapy and functional dressings in primary repair of spinal cord injury complicated with lacunar soft tissue defects: a comparison of efficacies
Jie YANG ; Xi YU ; Qian WANG ; Hongchen HE ; Hongying JIANG
Chinese Journal of Trauma 2023;39(10):925-932
Objective:To compare the efficacies of negative pressure wound therapy (NPWT) and functional dressings in primary repair of spinal cord injury complicated with lacunar soft tissue defects.Methods:A retrospective cohort study was conducted to analyze the clinical data of 30 patients with spinal cord injury complicated with lacunar soft tissue defects. The patients were admitted to West China Hospital, Sichuan University from January 2020 to December 2022, including 20 males and 10 females; aged 23-54 years [(42.1±7.8)years]. Wound site was located at the sacrococcygeal region in 16 patients, the buttock in 11, and the femoral trochanter in 3. Wound area was 28-36 cm 2 [(32.1±2.1)cm 2]. Time of wound formation was at range of 1-4 months [(2.0±0.8)months]. Among them, 15 patients received functional dressing treatment after mechanical/ultrasonic debridement (dressing treatment group), and 15 patients received NPWT treatment on the basis of mechanical/ultrasonic debridement (negative pressure treatment group). The following items were compared between the two groups: the time of primary wound repair, results of bacterial culture of wound secretions before and at the end of primary wound repair, and levels of serum interleukin-6 (IL-6) and C-reactive protein (CRP) as well as Bates-Jensen wound assessment tool (BWAT) score before, at 5 days after the primary repair and at the end of the primary repair. Results:All the patients were followed up for 3-6 months [(4.1±0.9)months]. The time of primary wound repair in the negative pressure treatment group was (13.4±2.3)days, which was markedly shorter than that in the dressing treatment group [(22.8±2.5)days] ( P<0.01). Before the primary repair, 11 patients in the negative pressure treatment group showed positive bacterial culture of wound secretions [73.3% (11/15)], and 9 patients in the dressing treatment group were positive [60.0% (9/15)] ( P>0.05). At the end of primary repair, there was 1 patient with positive bacterial culture of wound secretions in the negative pressure treatment group [6.7% (1/15)], which was less than 7 patients in the dressing treatment group [46.7% (7/15)] ( P<0.05). The numbers of positive patients at the end of the primary repair were lower than those before the primary repair in both groups, and the difference in the negative pressure treatment group was statistically significant ( P<0.01), with no significant difference found in the dressing treatment group ( P>0.05). Before the primary repair, the IL-6, CRP and BWAT score were 20.5(8.4, 32.3)pg/ml, 24.2(14.7, 33.0)mg/L, and (37.1±4.8)points in the negative pressure treatment group, comparable with 13.8(11.8, 35.4)pg/ml, 23.6(13.1, 52.3)mg/L, and (35.2±4.7)points in the dressing treatment group (all P>0.05). At 5 days after primary repair, the IL-6, CRP and BWAT score in the negative pressure treatment group were 20.2(7.9, 28.6)pg/ml, 20.0(11.6, 30.5)mg/L, and (34.9±4.3)points, comparable with 11.6(8.9, 20.6)pg/ml, 25.3(10.0, 50.3)mg/L, and (35.2±4.5)points in the dressing treatment group (all P>0.05). At the end of primary repair, the IL-6, CRP and BWAT score were 2.3(1.5, 4.5)pg/ml, 4.8(3.7, 6.9)mg/L, and (23.6±1.8)points in the negative pressure treatment group, statistically different from 4.4(3.3, 6.9)pg/ml, 8.4(5.5, 31.4)mg/L, and (31.4±3.3)points in the dressing treatment group (all P<0.01). The IL-6, CRP and BWAT score at the end of the primary repair were significantly different compared with those before and at 5 days after the primary repair in the two groups ( P<0.05 or 0.01). However, no significant difference was found between the two groups before and at 5 days after the primary repair (all P>0.05). Conclusion:Compared with functional dressings, NPWT can shorten the time required for primary repair of spinal cord injury complicated with lacunar soft tissue defects, control the inflammatory state of the wound, improve the trend of wound healing, and create a good condition for secondary repair treatment of the wound.
10.Clinical expert consensus on platelet-rich plasma treatment for lateral epicondylitis (2022 version)
Jian LI ; Guoqing CUI ; Chengqi HE ; Shiyi CHEN ; Boxu CHEN ; Hong CHEN ; Xuesong DAI ; Hongchen HE ; Hui KANG ; Tieshan LI ; Guoping LI ; Jiuzhou LU ; Chao MA ; Xin TANG ; Jun TAO ; Hong WANG ; Ming XIANG ; Dan XING ; Yiquan XIONG ; Qingyun XUE ; Rui YANG ; Tin YUAN ; Qiang ZHANG ; Jingbin ZHOU ; Weihong ZHU ; Yan XIONG ; Yan LIU
Chinese Journal of Trauma 2022;38(8):673-680
Lateral epicondylitis is a common clinical disease with characteristics of lateral elbow pain, insidious onset and easy recurrence, which can cause forearm pain and decreased wrist strength, seriously affecting patients′ daily life and work. Although there are various treatment methods for lateral epicondylitis with different effects, standard treatments are still lacking nowadays. Platelet-rich plasma (PRP) has good effects on bone and tendon repair, and is now widely used in the treatment of lateral epicondylitis. However, there is a lack of a unified understanding of the technology and specifications of PRP in the treatment of lateral epicondylitis. Therefore, the Sports Medicine Branch of the Chinese Medical Association and Physical Medicine and Rehabilitation Branch of the Chinese Medical Association organized experts in the fields of sports medicine and rehabilitation medicine in China to formulate the "clinical expert consensus on platelet-rich plasma treatment for lateral epicondylitis (2022 version)", and proposed suggestions based on evidence-based medicine mainly from the concept, epidemiology and pathophysiology of lateral epicondylitis, symptoms, signs and imaging manifestations of lateral epicondylitis, PRP concept and application component requirements, quality control of PRP preparation technology, indications and contraindications of PRP in the treatment of lateral epicondylitis, PRP injection in the treatment of lateral epicondylitis, application of PRP in the operation of lateral epicondylitis, related problems after PRP treatment of lateral epicondylitis, evaluation of the results after PRP treatment of lateral epicondylitis, and health and economic evaluation of PRP treatment of lateral epicondylitis, so as to provide guidance for clinical diagnosis and treatment.

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