1. Involvement of intracellular organelle stress, autophagy and ferroptosis in cobalt chloride-induced vascular smooth muscle cell injury
Yan LEI ; Xiaoyong PENG ; Tao LI ; Liangming LIU ; Mengsheng DENG ; Dongdong ZHANG ; Yingguo ZHU ; Jianmin WANG ; Zhaoxia DUAN ; Guangming YANG
Chinese Journal of Clinical Pharmacology and Therapeutics 2024;29(1):1-10
AIM: To investigate the relationship between vascular smooth muscle cell (VSMC) injury, organelle stress response and autophagic cell death (autophagy) and ferroptosis induced by the chemical hypoxia inducer cobalt chloride (CoCl2) through the bioinformatics analysis and in vitro cell experimentation. METHODS: The dataset GSE119226 of VSMC treated with cobalt chloride was acquired from the gene expression database (GEO). The R language was used to investigate the relationship between CoCl2 treatment and organelle stress response (Golgi stress, endoplasmic reticulum stress) and two forms of cell death (ferroptosis and autophagic cell death). With primary cultured rat VSMC (rVSMC) and CoCl2-induced anoxia model, the changes in cell viability were detected by CCK-8 method, and reactive oxygen species (ROS) levels were measured using DCFH-DA method. The expression levels of HIF-1α (a key molecule in hypoxia), Golgi stress markers GM130 and p115, endoplasmic reticulum stress markers GRP78 and CHOP, autophagy markers LC3-II / LC3-I and Beclin1, and ferroptosis markers GPx4 and xCT were detected by Western blot. The effect of inducing or inhibiting organelle stress and cell death on the CoCl2-induced cell damage was also observed. RESULTS: Differentially expressed genes analysis of GSE119226 dataset showed that CoCl2 treatment of VSMCs had significant effects on organelle function and stress response, autophagy and ferroptosis-related genes, in which endoplasmic reticulum stress, protein processing in endoplasmic reticulum, regulation of Golgi to plasma membrane protein transport, autophagy / autophagic cell death, and ferroptosis pathways were remarkably enriched. The results of in vitro experiment showed that compared with normal rVSMC, cell viability was significantly decreased after CoCl2 treatment, as well as HIF-1α protein expression and ROS levels in rVSMCs were increased. In rVSMC treated with Co-Cl2, the expression levels of Golgi structural proteins GM130 and p115 (reflecting the occurrence of Golgi stress) were decreased, while the markers GRP78 and CHOP (reflecting the occurrence of endoplasmic reticulum stress) were increased. At the same time, CoCl2 treatment also reduced the expression of autophagy markers LC3-II/LC3-I and Beclin1 (indicating the decrease levels of autophagy), while the expression of ferroptosis markers GPx4 and xCT were decreased (indicating the occurrence of ferroptosis). Compared with CoCl2 treatment group, induced Golgi stress, endoplasmic reticulum stress, or ferroptosis could further reduce cell viability, while inhibition of these processes could improve cell viability. On the other hand, increasing the level of autophagy can improve the cell viability. CONCLUSION: Hypoxia induced by cobalt chloride can lead to VSMC injury. Golgi stress, endoplasmic reticulum stress, ferroptosis, and the reduction of autophagy level play an important role in it. Inhibition of organelle stress response and ferroptosis, or increase of autophagy level can improve VSMC injury caused by cobalt chloride.
2.Negative association between composite dietary antioxidant index and excess heart age
ZHANG Wanyao ; HUANG Dongdong ; LU Ming ; REN Yong ; ZHU Tongjian ; LI Bin
China Tropical Medicine 2024;24(2):150-
Objective To investigate the relationship between the composite dietary antioxidant index (CDAI) and excess heart age (EHA), offering theoretical insights into the nutritional prevention of cardiovascular disease (CVD). Methods Data for this cross-sectional analysis was derived from the National Health and Nutrition Examination Survey (NHANES) from 2007 to 2020, which included 16 442 participants aged 30 to 74 years without a history of CVD. The heart age was estimated using the Framingham risk score function, and EHA was subsequently calculated. The CDAI was formulated based on intake levels of vitamins A, C, E, zinc, selenium, and carotenoids to represent the overall level of antioxidants. The association between CDAI and EHA was evaluated using multivariate linear regression, multivariate logistic regression, and restricted cubic splines (RCS). Results Multiple regression analysis indicated that CDAI was negatively correlated with both EHA (β=-0.07, 95%CI: -0.14 to -0.01) and high CVD risk (defined as EHA≥10 years) (OR=0.98, 95%CI: 0.96 to 1.00), with RCS analysis suggesting that this relationship is a nonlinear and inverse L-shaped. In the female subgroup, significant negative, nonlinear associations were observed between CDAI and both EHA (β=-0.10, 95%CI: -0.12 to 0.00) and high CVD risk (OR=0.97, 95%CI: 0.94 to 0.99). Conversely, in males, these correlations did not reach statistical significance. Conclusions Our findings suggest that individuals with higher CDAI compliance had a lower risk of CVD, and this negative correlation was gender specific. This discovery offers new theoretical guidance for the nutritional prevention of CVD, underlining the necessity for more comprehensive clinical and foundational research to further explore this association.
3.Expert consensus on odontogenic maxillary sinusitis multi-disciplinary treatment
Lin JIANG ; Wang CHENGSHUO ; Wang XIANGDONG ; Chen FAMING ; Zhang WEI ; Sun HONGCHEN ; Yan FUHUA ; Pan YAPING ; Zhu DONGDONG ; Yang QINTAI ; Ge SHAOHUA ; Sun YAO ; Wang KUIJI ; Zhang YUAN ; Xian MU ; Zheng MING ; Mo ANCHUN ; Xu XIN ; Wang HANGUO ; Zhou XUEDONG ; Zhang LUO
International Journal of Oral Science 2024;16(1):1-14
Odontogenic maxillary sinusitis(OMS)is a subtype of maxillary sinusitis(MS).It is actually inflammation of the maxillary sinus that secondary to adjacent infectious maxillary dental lesion.Due to the lack of unique clinical features,OMS is difficult to distinguish from other types of rhinosinusitis.Besides,the characteristic infectious pathogeny of OMS makes it is resistant to conventional therapies of rhinosinusitis.Its current diagnosis and treatment are thus facing great difficulties.The multi-disciplinary cooperation between otolaryngologists and dentists is absolutely urgent to settle these questions and to acquire standardized diagnostic and treatment regimen for OMS.However,this disease has actually received little attention and has been underrepresented by relatively low publication volume and quality.Based on systematically reviewed literature and practical experiences of expert members,our consensus focuses on characteristics,symptoms,classification and diagnosis of OMS,and further put forward multi-disciplinary treatment decisions for OMS,as well as the common treatment complications and relative managements.This consensus aims to increase attention to OMS,and optimize the clinical diagnosis and decision-making of OMS,which finally provides evidence-based options for OMS clinical management.
4.An analysis of genetic polymorphism and mutation rate of 36 Y-STR loci in Sichuan Han population
Shilin ZHANG ; Wei WANG ; Xiao ZHANG ; Wenli SHI ; Yanjie DING ; Dongdong XU ; Xiaohua ZHU ; Bo JIN
Chinese Journal of Forensic Medicine 2024;39(5):565-571,583
Objective To explore the genetic polymorphism and mutation rate of 36 Y-STR loci in Sichuan Han population and provide basic data for their forensic applications.Methods Blood samples were collected from 307 unrelated male individuals and 367 father-son pairs in Sichuan Han population using FTA blood sampling cards.The Y41SE-v1.2 kit was used to type 36 Y-STR loci,and the population genetic parameters and mutation rates of each locus were calculated.The genetic relationship between Sichuan Han population and other ethnic populations was analyzed in combination with 19 other populations in China.Results A total of 79 mutations were observed in 36 Y-STR loci in 367 father-son pairs of Sichuan Han population,with an average mutation rate of 6.00×10-3.There were 74(93.67%)one-step mutations and 5(6.33%)multi-step mutations.A total of 383 alleles were found at 36 Y-STR loci in 307 Sichuan Han males.The number of alleles at each locus ranged from 5(0.016 29)to 56(0.182 41),GD ranged from 0.100 02(DYS645)to 0.959 99(DYS385),and 306 haplotypes were found.The HD,DC and HMP values were 0.999 973,99.67%and 0.003 284,respectively.The results of genetic distance(Fst)analysis showed that the Sichuan Han population had the closest genetic distance with the Chongqing Han population(0.000 9)and the furthest genetic distance with the Xinjiang Mongolian group(0.032 0).The MDS based on genetic distance was basically consistent with the results of cluster analysis.Conclusion The 36 Y-STR loci have high genetic polymorphism in the Sichuan Han population.The study data can provide data support for the construction of the Y-STR database in this region,and also provide basic genetic information for forensic investigation and population genetics research.
5.Analysis of the diagnostic value of ultrasound elastography combined with EZH2 and BRCA1 proteins in triple-negative breast cancer
Jing YANG ; Juanying ZHU ; Ying ZHAO ; Li LING ; Xiaoping ZHU ; Dongdong CHEN
Chinese Journal of Endocrine Surgery 2023;17(5):545-549
Objective:To investigate the diagnostic value of ultrasound elastography combined with enhancer of Zeste homolog 2 (EZH2) and breast cancer susceptibility gene 1 (BRCA1) proteins in triple-negative breast cancer.Methods:A total of 116 breast cancer patients selected for treatment in our hospital from 2019 to 2021 were analyzed, and ER and PR were analyzed according to the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). Patients who were all HER2 negative were classified as TNBC group, with a total of 21 cases, and patients who were positive for ER, PR, and HER2 were classified as non-TNBC, a total of 95 cases. All enrolled patients underwent ultrasonic elastography examination. The positive expression of EZH2 and BRCA1 was detected by immunohistochemistry. Chi-square test was used to analyze the diagnostic value of ultrasound elastography combined with EZH2 and BRCA1 proteins in TNBC.Results:In the TNBC group, the proportion of smooth edges (35.09) and lobulation of lesions (9.52) was higher than that in the non-TNBC group (12.63, 5.26), while the proportion of burrs (28.57) and fuzziness (23.81) was lower than that in the non-TNBC group (50.53, 31.58). The proportion of calcifications (52.38) in the TNBC group was lower than that in the non-TNBC group (85.26). The non-attenuation ratio of rear echo (80.95) was higher than that of non-TNBC group (52.63). The proportion of blood flow grade enrichment (57.14) was lower than that in the non-TNBC group (63.16) (all P<0.05). The positive expression rate of EZH2 in the TNBC group (85.71) was higher than that of non-TNBC (63.16) ( P<0.001), and the positive expression rate of BRCA1 in the TNBC group (19.05) was lower than that of non-TNBC (55.79) ( P<0.05). The posterior echo non-attenuation ratio (88.89) and rich blood supply ratio (88.89) of EZH2 positive patients in the TNBC group were higher than those with negative (33.33, 0) (all P<0.05), and the rear echo non-attenuation ratio (25.00) and rich blood supply ratio (25.00) of BRCA1 positive in the TNBC group were lower than those with negative (52.94, 88.24) (all P<0.05) ; According to the pathological results, the sensitivity, specificity and accuracy of posterior echo for the diagnosis of TNBC were 25.37%, 91.84% and 53.44%, respectively. The sensitivity, specificity and accuracy of EZH2 were 23.08%, 92.11% and 45.69%, respectively. The sensitivity, specificity and accuracy of BRCA1 were 28.81%, 92.98% and 60.34%, respectively. The combined sensitivity, specificity and accuracy of the three were 53.13%, 95.24% and 83.62%, respectively. The sensitivity, specificity and accuracy of blood flow fraction for the diagnosis of TNBC were 25.81%, 90.74% and 56.03%, respectively. The sensitivity, specificity and accuracy of blood flow fraction, EZH2 and BRCA1 were 59.26%, 94.38% and 86.21%, respectively. Conclusion:The joint classification of the posterior echoes and blood flow in ultrasonic elastography combined with EZH2 and BRCA1 has important significance in the diagnosis of TNBC.
6.Effects of high-energy shock and vibration on cortex and peripheral blood immune cells in goats
Yingguo ZHU ; Dongdong ZHANG ; Liangchao ZHANG ; Guanhua LI ; Ling GUO ; Zhaoxia DUAN ; Jing CHEN ; Jianmin WANG ; Guangming YANG
Chinese Journal of Trauma 2023;39(11):1029-1036
Objective:To investigate the effects of high-energy shock and vibration on cortex injury and peripheral blood immune cells in goats.Methods:Seventeen Boer goats without gender preference were selected. By using random number tables, the goats were divided into normal control group ( n=5) and shock and vibration injury group ( n=12). The goats in the normal control group were anatomized routinely and their brain was collected after being sacrificed without any other treatment. The goats in the high-energy shock and vibration model group were placed on a loading table (part of the BY10-100 instant shock and vibration simulation platform) in a restrained state, and made into a high-energy shock and vibration injury model induced by a vertical impact waveform generator. The intravenous blood samples were taken from the goats in the shock and vibration injury group before and at 0, 3, 6 and 24 hours after injury.Then, the goats were sacrificed and the following procedures were the same as the normal control group. At 24 hours after injury, the brain injury and the histopathological changes of the cerebral cortex in the normal control group and shock and vibration injury group were observed by gross pathological and anatomical examination and HE staining. The mRNA expression of zonula occludens 1 (ZO-1), tight junction protein 5 (Claudin-5), glial fibrillary acidic protein (GFAP), ionized calcium binding adaptor molecule 1 (IBA-1), interleukin (IL)-1β, IL-6 and cluster of differentiation antigen 177 (CD177) of the cerebral cortex in the normal control group and shock and vibration injury group were measured through fluorogenic quantitative polymerase chain reaction. The expression of ZO-1 and Claudin-5 proteins of the cerebral cortex in the normal control group and shock and vibration injury group were detected by Western blotting. Hematology analyzer and coagulation analyzer were used to detect white blood cell count, neutrocyte, lymphocyte, monocyte, prothrombin time 1 (PT-1), prothrombin time international normalized ratio (PT-INR), activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin activity (PTA) and fibrinogen (FIB) levels in goats of the shock and vibration injury group before and at 0, 3, 6 and 24 hours after injury, respectively. Results:At 24 hours after injury, no visible contusion or necrosis was found in goat brain tissue in the shock and vibration injury group; the cerebral micro-vessels presented with a local dilation, hyperemia, edema, aggregation of inflammatory cells, disruption of vessel walls and leakage of red blood cells. These changes were not observed in the normal control group. In the shock and vibration injury group, ZO-1 and Claudin-5 mRNA expressions in the cerebral cortex were 0.25±0.10 and 0.09(0.04, 0.44) respectively, which were significantly lower than those of the normal control group [1.00±0.15 and 0.99(0.80, 1.20)]; GFAP, IBA-1, IL-1β, IL-6 and CD177 mRNA expression levels were 4.40(3.88, 6.75), 2.60±1.07, 3.04±0.51, 2.71±0.45 and 2.93±0.62 respectively, which were significantly higher than those of the normal control group [1.00(0.78, 1.22), 1.00±0.37, 1.00±0.27, 1.00±0.57 and 1.00±0.35]; ZO-1 and Claudin-5 protein expression levels were 0.41±0.06 and 0.42±0.11 respectively, which were significantly lower than those of the normal control group (1.08±0.12 and 0.91±0.23) (all P<0.01). In the shock and vibration injury group, the levels of white blood count, neutrocyte, and lymphocyte in peripheral blood were (13.7±3.3)×10 9/L, (35.3±14.8)% and (57.2±15.1)% respectively before injury, (19.4±3.1)×10 9/L, (60.5±12.5)% and (33.6±14.2)% respectively at 3 hours after injury, and (20.6±3.6)×10 9/L, (63.6±13.0)% and (30.9±15.0)% respectively at 6 hours after injury. By contrast, the levels of white blood count and neutrocyte were significantly increased but the level of lymphocyte was significantly decreased at 3 and 6 hours after injury ( P<0.05 or 0.01); the levels of the above indicators showed no significant changes at 0 and 24 hours after injury (all P>0.05); the level of monocyte did not change significantly at all time points before and after injury (all P>0.05). The levels of PT-1, PT-INR, APTT, TT, PTA and FIB in the shock and vibration injury group did not change significantly at each time point before and after injury (all P>0.05). Conclusion:Cerebral cortex microvascular injury and disruption of blood-brain barrier can be initiated in the early stage of high-energy shock and vibration injury in goats, accompanied by the presence of central and peripheral inflammatory response.
7.Role of endoplasmic reticulum stress apoptosis protein in cardiac injury induced by endostar plus irradiation in rats
Qiying CHEN ; Dongdong CAO ; Weiwei OUYANG ; Li HUANG ; Shengfa SU ; Zhu MA ; Qingsong LI ; Yichao GENG ; Wengang YANG ; Xiaxia CHEN ; Jie LIU ; Bing LU
Chinese Journal of Radiation Oncology 2023;32(10):920-927
Objective:To study the relationship between endoplasmic reticulum stress (ERS) and apoptotic protein and myocardial pathological changes in rats after endostar combined with low-dose X-ray irradiation.Methods:Forty SD rats were evenly divided into four groups: control group (intraperitoneal injection of equal volume physiological saline, once per day, 14 d), endostar group (intraperitoneal injection of endostar 6 mg/kg, once per day, 14 d), irradiation group (15 Gy divided into 3 times X-ray irradiation) and combination group (intraperitoneal injection of endostar after irradiation at the same dose and time as the endostar group). At 1 and 6 months after treatment, myocardial tissues of rats were prepared for HE staining and Masson staining to observe the myocardial histological changes. TUNEL assay was used to detect myocardial cell apoptosis, and ImageJ software was utilized to calculate myocardial collagen volume fraction (CVF). The expression levels of ERS and apoptotic protein glucose-regulated protein 78 (GRP78), protein kinase-like endoplasmic reticulum kinases (PERK), CCAAT/enhancer binding protein homologous protein (CHOP) and cysteine-containing aspartate-specific protease-12 (Caspase-12) were detected by Western blot. One-way ANOVA was conducted using GraphPad Prism 8.0.1 software, and comparison between two groups was conducted using t-test. Results:At 6 months after treatment, the myocardial interstitium in the irradiation and combination groups was widened, showing strip-like or reticular fibrosis changes, and the myocardial interstitium had diffuse collagen fiber deposition. Compared with the control group, CVF was increased significantly (both P<0.01). At 1 and 6 months after treatment, the apoptotic index of myocardial cells in the combination group was significantly higher than that in the control group ( P<0.05, <0.001). At 1 and 6 months after treatment, the expression levels of GRP78 protein in the irradiation and combination groups were increased (all P<0.01), and the expression levels of PERK and CHOP proteins in the combination group were increased compared to those in the control group (both P<0.05). At 6 months after treatment, the expression levels of PERK and CHOP proteins in the irradiation group were increased compared to those in the control group (both P<0.05). Compared with the control group, Caspase-12 expression levels at 1 and 6 months after treatment were increased in the endostar, irradiation and combination groups (all P<0.05). Conclusions:The expression levels of ERS and apoptotic proteins are related to cardiac injury caused by irradiation in rats. After low-dose X-ray combined with endostar treatment, ERS is aggravated and myocardial apoptosis is increased.
8.A multi-center observation of the therapeutic efficacy of Bencycloquidium bromide in the treatment of seasonal allergic rhinitis with predominant symptoms of rhinorrhea.
Weini HU ; Tianhong ZHANG ; Yinghong ZHANG ; Chao MENG ; Lifeng XIE ; Yu SONG ; Chen DU ; Chiyu XU ; Yali DU ; Qiang ZUO ; Fengyang AN ; Yuhui WANG ; Cuida MENG ; Lei ZHANG ; Dongdong ZHU ; Li ZHU
Journal of Clinical Otorhinolaryngology Head and Neck Surgery 2023;37(7):550-555
Objective:To observe the efficacy and safety of the M receptor antagonist Bencycloquidium bromide nasal spray in treatment of seasonal allergic rhinitis with runny nose as the main symptom. Methods:From August 2021 to September 2021, 134 patients with seasonal allergic rhinitis were enrolled in the otolaryngology Outpatient Department of Peking University Third Hospital, First Affiliated Hospital of Harbin Medical University and China-Japanese Friendship Hospital of Jilin University, including 71 males and 63 females, with a median age of 38 years. TNSS score and visual analogue scale(VAS) of total nasal symptoms were observed during 2 weeks of treatment with Bencycloquidium bromide nasal spray. Results:TNSS score decreased from (8.89±3.31) on day 0 to (3.71±2.51) on day 14(P<0.001), VAS score of nasal symptoms decreased from (24.86±7.40) on day 0 to (6.84±5.94) on day 14(P<0.001), VAS score of rhinorrhoea decreased from (6.88±2.06) on day 0 to (1.91±1.81) on day 14(P<0.001). Rhinoconjunctivitis quality of life questionnaire(RQLQ) score decreased from (94.63±33.35) on day 0 to (44.95±32.28) on day 14(P<0.001). The incidence of adverse reaction was low and no serious adverse events occurred during the whole experiment. Conclusion:Bencycloquidium bromide nasal spray has significant efficacy and good safety in the treatment of seasonal allergic rhinitis.
Male
;
Female
;
Humans
;
Adult
;
Rhinitis, Allergic, Seasonal/drug therapy*
;
Nasal Sprays
;
Quality of Life
;
Administration, Intranasal
;
Rhinorrhea
;
Double-Blind Method
;
Treatment Outcome
;
Rhinitis, Allergic/drug therapy*
9.Isolated metastasis of the ascending ramus of the mandible of thyroid follicular carcinoma: a case report.
Siyao ZHANG ; Qingjia SUN ; Dongdong ZHU
Journal of Clinical Otorhinolaryngology Head and Neck Surgery 2023;37(7):574-577
The mandibular metastatic spread of carcinoma from the thyroid gland is exceedingly rare. Follicular thyroid carcinoma is the second most common type of thyroid carcinoma,accounting for approximately 10% to 15% of all thyroid cancers. The prognosis of FTC is relatively satisfactory. Due to its rich blood transport, it is easy to metastasize hematological, with the main sites of metastasis are bone and lung. However,mandibular metastasis of thyroid follicular carcinoma is rare. We report a case of thyroid follicular carcinoma that metastasized to the ascending ramus of the mandible 21 years after surgery.The operation was successfully completed, and there was no recurrence during postoperative follow-up. Due to the absence of obvious clinical symptoms in the patient, the diagnosis and treatment were challenging. We have provided detailed radiographic and pathological images to facilitate understanding and discussion of the disease.
Humans
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Adenocarcinoma, Follicular/pathology*
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Thyroid Neoplasms/surgery*
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Prognosis
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Mandible
10.The role of miR-21 in Endostar combined with X-ray irradiation of cardiac fibroblasts
Dongdong CAO ; Weiwei OUYANG ; Xing ZHAO ; Shengfa SU ; Jun ZHANG ; Shimei FU ; Zhangxin FENG ; Qingsong LI ; Zhu MA ; Wengang YANG ; Xiaxia CHEN ; Bing LU
Chinese Journal of Radiation Oncology 2022;31(4):370-375
Objective:To investigate the implication of micro RNA-21(miR-21) in Endostar combined with X-ray irradiation of cardiac fibroblasts (CF).Methods:Rat CFs were used in this experiment and been divided into the blank control group, 10 Gy X-ray irradiation group, Endostar group, 10 Gy X-ray+ Endostar group, 10 Gy X-ray+ Endostar+ NC mimic group (negative control 1), 10 Gy X-ray+ Endostar+ miR-21 mimic group, 10 Gy X-ray+ Endostar+ NC inhibitor group (negative control 2) and 10 Gy X-ray+ Endostar+ miR-21 inhibitor group. The proliferation of CF was determined by Methyl thiazolyl tetrazolium (MTT) assay. The expression level of Collagen Ⅰ protein was analyzed by Western blot. The expression levels of Collagen Ⅰ and miR-21 mRNA were assayed by real-time quantitative polymerase chain reaction (q-PCR).Results:In the 10 Gy X-ray+ Endostar+ miR-21 mimic group, the CF proliferation, Collagen Ⅰ and miR-21 mRNA were increased significantly compared with those in the blank control group, 10 Gy X-ray+ Endostar group, and negative control group 1 (all P<0.05). In the 10 Gy X-ray+ Endostar+ miR-21 inhibitor group, the CF proliferation and expression levels of Collagen Ⅰ mRNA were decreased significantly compared with those in the blank control group, 10 Gy X-ray+ Endostar group and negative control group 2(all P<0.05). Conclusions:The CF proliferation and Collagen Ⅰ expression are increased when the expression level of miR-21 gene is simulated. When inhibiting the expression of miR-21 gene, the CF proliferation and Collagen Ⅰ expression are reduced.

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