The cartilage-protective effect of ginkgolide C(GC)on the two modeling modalities was investigated based on joint pain,degree of cartilage pathology,ECM degradation process,and level of inflammatory mediator production in rats.Twenty-five SD rats were selected and randomly di-vided into five groups:the control group(Control group),model 1 group(ACLT group),adminis-tration 1 group(ACLT+GC group),model 2 group(MIA group),and administration 2 group(MIA+GC group.)The rats were euthanized after 4 weeks of the test.Femur,tibia and blood samples were collected from the right hind limb of rats.The degree of pathology in the femur and tibia of rats was assessed by saffron O solid green staining and OARSI score.Immunohistochemis-try was used to detect the expression levels of collagen Ⅱ and MMP-13 in cartilage.ELISA was used to detect the changes in the levels of MMP-3,MMP-13,CTX-Ⅱ,COMP,COX-2,INOS,IL-1β,and TNF-α in the serum of rats.Cold sensitivity test and knee extension vocalization test were conducted to detect the degree of joint pain in rats.ACLT could cause more severe structural dam-age to articular cartilage compared with the MIA group.The OARSI scores and the expression of MMP-13 in femur and tibia,and the serum levels of MMP-13,MMP-3,CTX-Ⅱ,and COMP were higher in the ACLT group than those in the MIA group.However,the levels of inflammatory me-diators COX-2,IL-1β,and TNF-α were significantly lower in the ACLT group than in the MIA group(P＜0.0l).GC intervention reduced the OARSI score(P＜0.05 or P＜0.01)and pain scores,inhibited the ECM matrix degrading enzymes(MMP-13,MMP-3),cartilage metabolism markers(CTX-11,COMP),and inflammatory mediators(COX-2,INOS,IL-1β and TNF-α)ex-pression,and promoted collagen Ⅱ synthesis.Both modeling methods resulted in cartilage damage.In particular,the OA model constructed by ACLT+PMMx method in rats had obvious joint dam-age,which was favorable to investigate the degree of cartilage structural damage.GC attenuated cartilage pathological changes,pain severity and inflammatory response in the rat OA model in both groups,thus exerting a cartilage-protective effect.

Objective:To investigate the expression levels of miRNA-21 (miR-21) and miRNA-330 (miR-330) in serum exosomes of non-small cell lung cancer (NSCLC) patients with brain metastases, and the correlation of the two with the prognosis of patients.Methods:A prospective cohort study was conducted. A total of 125 NSCLC patients who were admitted to the Affiliated People's Hospital of Shandong First Medical University from March 2021 to September 2022 were prospectively selected, and the brain metastasis was determined by CT, contrast-enhanced magnetic resonance imaging of the head, or surgical pathology. The NSCLC patients were divided into the metastatic group (58 cases) and the non-metastatic group (67 cases) according to whether they had brain metastases, and 50 patients with benign lung diseases and 50 healthy subjects who underwent physical examination in the same period were selected as benign group and healthy control group respectively. Serum samples were collected from all subjects (including patients' pre-treatment samples), the exosomes were extracted, and real-time fluorescence quantitative polymerase chain reaction was used to determine the relative expression of miR-21 and miR-330 in exosomes at the transcriptional level, and electrochemiluminescence immunoassay was used to detect the levels of serum tumor markers [neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCCA)]. The levels of miR-21 and miR-330 in serum exosomes and serum tumor markers in the 4 groups were compared, and the correlation between miR-21 and miR-330 in serum exosomes of NSCLC patients with brain metastases before treatment and the correlation between miR-21, miR-330 and serum tumor markers were analyzed by Pearson method. Using brain metastases identified by CT, contrast-enhanced magnetic resonance imaging of the head or surgical pathology as the gold standard, the receiver operating characteristic (ROC) curves were drawn to determine the occurrence of brain metastases in NSCLC patients based on the levels of miR-21, miR-330 and their combination in the serum exosomes before treatment. NSCLC patients were divided into the poor prognosis group and the good prognosis group according to whether or not they died of tumor during the follow-up period, and the clinical characteristics and levels of miR-21 and miR-330 in serum exosomes before treatment were compared between the two groups. The independent influencing factors of death due to tumor in NSCLC patients were analyzed by multivariate logistic regression.Results:Among 125 NSCLC patients, 68 (54.4%) were male and 57 (45.6%) were female; the age was (63±5) years old, ranging from 49 to 82 years old; 89 patients (71.2%) were adenocarcinoma and 36 patients (28.8%) were squamous cell carcinoma. The transcriptional level relative expression of miR-21 in serum exosomes of healthy control group, benign group, non-metastatic group and metastatic group increased sequentially, the transcriptional level relative expression of miR-330 decreased sequentially, the protein concentrations of NSE, CEA and SCCA increased sequentially, and the differences between each two groups were statistically significant (all P<0.001). Pearson correlation analysis showed that in the serum exosomes of NSCLC patients with brain metastases before treatment, miR-21 was positively correlated with serum NSE, CEA and SCCA levels ( r values were 0.641, 0.785 and 0.612, respectively; P values were 0.015, 0.011 and 0.019, respectively), miR-330 in the serum exosomes before treatment was negatively correlated with serum NSE, CEA, and SCCA levels ( r values were -0.612, -0.689 and -0.587, respectively; P values were 0.016, 0.021 and 0.013, respectively), and miR-21 was positively correlated with miR-330 in the serum exosomes before treatment ( r = -0.529, P = 0.023). ROC curve analysis showed that the area under the curve of miR-21, miR-330 and their combination in serum exosomes before treatment for determining the occurrence of brain metastases in NSCLC patients were 0.861 (95% CI: 0.792-0.931), 0.894 (95% CI: 0.840-0.947) and 0.906 (95% CI: 0.849-0.963), and the differences were statistically significant (all P < 0.001). The optimal cut-off value of miR-21 relative expression was 1.625, and the corresponding sensitivity and specificity were 77.4% and 71.5%, respectively; the optimal cut-off value of miR-330 was 0.611, and the corresponding sensitivity and specificity were 81.1% and 74.9%, respectively; the sensitivity and specificity when the two were combined to reach the optimal cut-off value were 84.5% and 73.8%, respectively. NSCLC patients were followed up for a median time of 19 months (95% CI: 17-21 months), and 23 cases (18.4%) died due to the tumor during the follow-up period. The proportions of patients with age ≥60 years old, clinical stage Ⅲ-Ⅳ and brain metastases and the relative expression of miR-21 in serum exosomes before treatment in the poor prognosis group were higher than those in the good prognosis group, the relative expression of miR-330 in the serum exosomes before treatment was lower than that in the good prognosis group, and the differences were all statistically significant (all P < 0.05). Multivariate logistic regression analysis showed that the high age (≥60 years old vs. <60 years old, OR = 3.750, 95% CI: 1.191-11.806, P = 0.024), late clinical stage (stage Ⅲ-Ⅳ vs. stage Ⅰ-Ⅱ, OR = 4.667, 95% CI: 1.303-16.716, P = 0.018), brain metastasis (with metastasis vs. non-metastasis, OR = 2.573, 95% CI: 1.008-6.611, P = 0.049), and elevated relative expression of miR-21 in serum exosomes before treatment ( OR = 2.585, 95% CI: 1.198-6.152, P = 0.008) were the independent risk factors for death due to tumor in NSCLC patients, and elevated relative expression of miR-330 in serum exosomes before treatment was an independent protective factor for death due to tumor ( OR = 0.821, 95% CI: 0.715-0.954, P < 0.001). Conclusions:miR-21 level is high and miR-330 level is low in serum exosomes of NSCLC patients with brain metastases before treatment, and there is a negative correlation between them, and they are closely related to various serum tumor markers of NSCLC patients with brain metastases and NSCLC patients' prognosis; the combination of the two may predict the occurrence status of brain metastases in NSCLC.

Early detection and intervention are key strategies to reduce mortality, increase long-term survival, and improve the therapeutic effects of hepatocellular carcinoma (HCC) patients. Herein, the isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative pro-teomic strategy was used to study the secretomes in conditioned media from HCC cancerous tissues, surrounding noncancerous tissues, and distal noncancerous tissues to identify diagnostic and prog-nostic biomarkers for HCC. In total, 22 and 49 dysregulated secretory proteins were identified in the cancerous and surrounding noncancerous tissues, respectively, compared with the distal non-cancerous tissues. Among these proteins, carbonic anhydrase Ⅱ (CA2) was identified to be signifi-cantly upregulated in the secretome of cancerous tissues; correspondingly, the serum concentrations of CA2 were remarkably increased in HCC patients compared with that in normal populations. Interestingly, a significant increase of serum CA2 in recurrent HCC patients after rad-ical resection was also confirmed compared with HCC patients without recurrence, and the serum level of CA2 could act as an independent prognostic factor for time to recurrence and overall sur-vival. Regarding the mechanism, the secreted CA2 enhances the migration and invasion of HCC cells by activating the epithelial mesenchymal transition pathway. Taken together, this study identi-fied a novel biomarker for HCC diagnosis and prognosis, and provided a valuable resource of HCC secretome for investigating serological biomarkers.

Objective To explore the value of contrast?enhanced T1 mapping technique in differentiating between recurrence and radiation necrosis of brain metastases after gamma knife treatment. Methods From March 2016 to June 2017,56 patients with brain metastases treated by gamma knife and confirmed by pathology or follow?up in Shandong Provincial Hospital were prospectively collected. Routine MRI and contrast?enhanced T1 mapping sequence scan were performed. T1 value was obtained 5 mins (T1 5 min) and 60 mins (T1 60 min) after injection of contrast agent. The Differences betweenT1 60 min and T1 5 min (T1 differ) was calculated,and relative cerebral blood volume (rCBV) value was obtained. Patients were divided into radiation necrosis group and tumor recurrence group according to imaging follow?up results or pathological results. Two?sides unpaired t test was used to compare the differences in T1 5 min,T1 60 min,T1 differ and rCBV between the 2 groups. Pearson correlation analysis was used to evaluate the correlation between T1 differ and rCBV, and the receiving operating curve (ROC) was used to evaluate the diagnostic efficiency of MRI quantitative parameters,and Z test was used to compare the differences of area under curve (AUC) between T1 differ and rCBV values. Results Of the 56 patients,27 had tumor recurrence and 29 had radiation necrosis. The differences in T1 5 min,T1 60 min,T1 differ and rCBV between the 2 groups was statistically significant (P<0.05). T1 differ and rCBV values were significantly correlated, r=0.58, P<0.01. The differential diagnosis of AUC between radiation necrosis and tumor recurrence were 0.66,0.73,0.97 and 0.95 respectively in T1 5 min,T1 60 min, T1 differ and rCBV, and there was no significant differences between AUC in T1 differ and rCBV (P=0.274). Conclusion The contrast?enhanced T1 mapping image can be used for differential diagnosis between radiation necrosis and recurrence after gamma knife treatment of brain metastases. T1 differ value has high differential efficiency.

Objective To study the association between CYP2C9 gene polymorphism and warfarin maintenance dosage in anticoagulation therapy.Methods 200 Han patients admitted to our hospital for heart valve replacement were included in this study.CYP2C9 * 2,CYP2C9 * 3,CYP2C9 *c65 in CYP2C9 gene were sequenced using the CAPS technique and conventional DNA sequencing method.Dosages of warfarin used in patients carrying different genes were analyzed.Results No mutation of CYP2C9 * 2 but only one kind of allele C was detected in 200 patients.The genotype of CYP2C9 * 2 was C/C wild type.Allelic gene was detected at CYP2C9 * 3 A and C,with A/A wild type detected in 171 patients,A/C heterozygote mutation type detected in 18 patients,and C/C heterozygote mutation type detected in 11 patients respectively.The frequency of allelic genes A and B was 94.3 ％ and 5.7 ％ respectively.A significant difference was found between CYP2C9 * 3 mutation and warfarin dosage (P＜0.05).The dosage of warfarin reduced 18.46％ and 76.0％ respectively in patients carrying A/C heterozygote mutation type and in those carrying C/C heterozygote mutation type.Two kinds of allelic gene were detected at CYP2C9 * c65 G and C,with G/G wild type detected in 182 patients and G/C heterozygote mutation type detected in 18 patients respectively.No significant association was found in warfarin maintenance dosage for patients carrying G/G wild type and G/C heterozygote mutation type.Conclusion CYP2C9 gene polymorphism is associated with warfarin maintenance dosage in anticoagulation therapy.

This paper was aimed to observe the effect of Yisui Shengxue (YSSX) granules on CD4+ CD25 + regulatory T cells (Treg cells) and its treatment mechanism in aplastic anemia (AA) rats.Male SD rats were selected and randomly divided into different groups according to their weight.In the model group,subcutaneous injection of benzene (1 mL· kg-1)was given every other day for 7 consecutive weeks.Ten days before the rats were sacrificed,intraperitoneal injection of CTX (25 mL · kg-1) was given for 3 consecutive days.On the 4th week,model rats were divided into the model group,stanozolol group,and the YSSX granules group.Intragastric administration of corresponding drug was given.Same volume of normal saline was given to the normal group and the model group.At the end of the experiment,WBC,RBC,HGB and PLT in peripheral blood were detected.Blood smear and bone marrow smear were prepared.The Foxp3 protein expression of Treg cells in spleen tissues was detected by immunohistochemistry (IHC).RT-PCR was used to detect the Foxp3 mRNA expression in bone marrow tissues.The results showed that compared with the normal group,WBC,RBC,HGB and PLT in the model group were significantly reduced (P ＜ 0.01).The blood smear showed poor permeability of blood cells,reduced WBCs,and increased degenerated cells.The bone marrow smear indicated significantly increased fat drops,significantly reduced hematopoietic cells,and increased nonhematopoietic cells.After the treatment of YSSX granules,WBC,RBC,HGB and PLT were significantly increased (P ＜ 0.01).Both the blood smear and bone marrow smear showed cell permeability improvement,cell form returns to normal,fat drops significantly reduced,significantly increased hematopoietic cells,significantly increased Foxp3 protein expression in spleen tissues and Foxp3 mRNA expression in bone marrow tissues (P ＜ 0.01).It was concluded that YSSX granules can upregulate both gene and protein expression of Foxp3,regulate AA immune function in order to improve the AA immune environment,promote the recovery of bone marrow hematopoietic function,which played an important role in AA treatment.

Objective To introduce a new method of trans-superior limb of cerebellopontine fissure approach for exploring and managing superomedial responsible vessels of facial nerve of patients with hemifacial spasm.Methods Clinical data of 21 patients with hemifacial spasm among 183 consecutive patients were analyzed retrospectively in our hospital from February 2009 to December 2013.Dissection of the superior limb of the cerebellopontine fissure was performed to explore the distribution and the severity of compression of the superomedial responsible vessels of the facial nerve,and microvascular decompression was performed.Results Neurovascular compression was found in all of the patients,primary responsible vessels were found in 13 patients,and secondary responsible vessels were found in 8 patients.Complete spasm alleviation was achieved immediately after operation in 18 patients,and complete spasm alleviation was achieved in all of the patients 3 months after operation.No severn complications occurred and no patient died.No recurrence was noted after an average 56 months of follow-up.Conclusion The trants-superior limb of cerebellopontine fissure approach could avoid the defects of standard suboccipital retrosigrnoidal approach,which allows easy identification and management of the superomedial responsible vessels of the facial nerve of patients with hemifacial spasm;thus,high consistent successful rate and low complication rate could be found.

Objective To compare the planning quality and volume of organ at risk (OAR) between volumetric-modulated arc therapyv (VMAT) and nine-field dynamic intensity-modulated radiation therapy (IMRT) in radiotherapy for cervical cancer patients,explore the best way to cervical cancer radiotherapy,Methods Selected 20 patients with cervical cancer were divided into 2 groups,10 cases for each group.Cervical cancer patients with no surgery was performed for A group (group A),received the radical radiotherapy,prescription dose gross tumor volume (GTV) 56 Gy,clinical target volume (CTV) 50 Gy.Another group of patients with cervical cancer radical surgery (group B),giving the whole basin lymph node auxiliary radiation therapy,prescription dose CTV 50 Gy.Each cervical cancer patient received VMAT and IMRT program designs,the differences in dose volume histogram (DVH),irradiated volume of organ at risk (OAR),heterogeneity index (HI),conformity index (CI),maximum dose (PTVmax),minimum dose (PTVmin) and mean dose (PTV mean) were compared between two plans in 2 groups.Results Two kinds of radiation technology in target area dosimetry were not statistical difference between two groups (P ＞ 0.05).In endanger organs on the protection of two groups of VMAT planning groups in the small intestine V20 and left femoral head V20 had obvious advantages with statistically significance (P ＜ 0.05).Conclusions Two groups of dosimetry between VMAT and IMRT program design are similar in cervical cancer.Two groups of VMAT planning groups to protect endanger organ slightly better than that of IMRT group,but VMAT planning group shortens treatment time and improves the accuracy and efficacy of treatment.

This study was aimed to clone the GGPS (geranylgeranyl pyrophosphate synthase) gene from Lepidium apetalum, to analyze its sequence, and to express the protein in E.coli expression system. Specific PCR cloning primers were designed for GGPS gene from Lepidium apetalum according to the full-length sequence from a previous transcriptome sequencing project. PCR amplification was performed with this primer pair on a leaf cDNA template. TA cloning, sequencing and sequence analysis were performed.GGPS gene from Lepidium apetalum was expressed in the E.coli expression system. The results showed that the full-lengthGGPS cDNA from Lepidium apetalum was 1 146 bp coding a protein of 381 amino acids. The LaGGPS protein had an isoprenoid synthase domain. According to a phylogenetic tree constructed with multiple alignment of GGPS protein sequences from various plant species, GGPS protein from Lepidium apetalum was the closest to Arabidopsis thaliana and Sinapis alba. The prokaryotic expression vectorpET-32a-LaGGPS was also constructed successfully. The protein was expressed in E.coli BL21 strain. It was concluded that the cloning and prokaryotic expression of LaGGPS gene provided a foundation for a follow-up research of its function with protein purification and activity analysis.

OBJECTIVETo explore significance of serum soluble CD163(sCD163) and soluble CD25(sCD25) in diagnosis and guiding treatment of children with hemophagocytic lymphohistiocytosis (HLH).

METHODData of 42 cases of children with HLH, 32 cases of non-HLH children with infection presented to First Affiliated Hospital of Zhengzhou University pediatric clinic and ward were collected from December 2013 to December 2014. Twenty-four healthy children were enrolled into a normal control group in the same period.Peripheral venous blood specimens (3 ml) were taken from the children with HLH after fasting before treatment, two weeks after treatment and eight weeks after treatment.Peripheral venous blood specimens (3 ml) were also taken from children of non-HLH infected group and normal control group after fasting at the initial visit. Serum sCD163 and sCD25 levels in the peripheral blood in three groups were determined by ELISA. According to cause of disease, children with HLH were divided into infection-related HLH, tumor-related HLH, primary HLH and others; relationship between serum sCD163 and sCD25 level and cause of disease was analyzed.

RESULTSerum sCD163 of HLH group ((6 094 ± 2 769) µg/L) and serum sCD163 of non-HLH infection group ((2 174 ± 950) µg/L) were significantly higher than that of normal control group ((777 ± 256) µg/L), F=71.396, P<0.05), and the differences among groups were statistically significant (P<0.05); serum sCD25 of HLH group ((41 963 ± 31 821) ng/L) and serum sCD25 of non-HLH infection group ((6 700 ± 4 105) ng/L) were significantly higher than that of normal control group ((2 440 ± 1 870) ng/L, F=37.513, P<0.05).There was no statistically significant difference between the non-HLH infection group with the normal control group (P>0.05), and the difference between the remaining groups was statistically significant (P<0.05). And serum sCD163 and sCD25 level of HLH group had a positive linear correlation, and Pearson correlation coefficient r=0.742 (t=7.000, P<0.05). The difference of serum sCD163 and sCD25 level among the different cause of disease in HLH group was significant (P<0.05).Pairwise comparison showed that serum sCD163 and sCD25 level of tumor-associated HLH group significantly increased as compared with infection-associated HLH group (P<0.05), but the difference was not statistically significant between the other groups (all P>0.05). Serum sCD163 and sCD25 level of HLH group before treatment, 2 weeks and 8 weeks after treatment showed a statistically significant tendency of decrease (P<0.05). Seen from the ROC curve, when sCD163 cut-off point was 2 359.08 µg/L, the diagnostic sensitivity was 83.3%, and specificity was 83.9%.When sCD25 cut-off point was 14 901.024 ng/L, the diagnosis sensitivity was 76.2%, and specificity was 98.2%.

CONCLUSIONSerum sCD163 and sCD25 levels may be used for diagnosis of HLH.Dynamically monitoring of serum sCD163 and sCD25 level can help to determine deterioration of HLH and guide treatment.

Antigens, CD ; blood ; Antigens, Differentiation, Myelomonocytic ; blood ; Case-Control Studies ; Child ; Enzyme-Linked Immunosorbent Assay ; Humans ; Interleukin-2 Receptor alpha Subunit ; blood ; Lymphohistiocytosis, Hemophagocytic ; blood ; diagnosis ; therapy ; ROC Curve ; Receptors, Cell Surface ; blood ; Sensitivity and Specificity