Objective To observe the biological activity of the recombinant immunotoxin hIL-2-LuffinP1 derived by gene engineering in vitro.Methods To observe the inhibitory effect on lymphocytes in vitro,the splenocytes isolated from C57 and BALB/c mice were used for mixed mouse lymphocyte response(MLR),and the lymphocyte proliferation was observed in vitro with BALB/c mice splenocytes stimulated by concanavalin A(ConA).Different concentrations of hIL-2-LuffinP1 was added into the reaction system,with LuffinP1 adopted as control.3H-TdR incorporation assay was used to detect the effects of hIL-2-LuffinP1 on lymphocyte proliferation,and its effects on CTLL-2 cells(IL-2 dependent cells) were also observed.The experiments consisted of six groups(PBS,IL-2,LuffinP1,LuffinP1+IL-2,hIL-2-LuffinP1 and hIL-2-LuffinP1+IL-2).CTLL-2 cells were cultured for 12h after being treated with different agents,and then the cells were stained by trypan blue to estimate the dead cells in every 100 cells.Results It was showed that CPM declined correspondingly to the gradually increased concentration of hIL-2-LuffinP1,and cell proliferation was inhibited obviously.The inhibition rate was up to 97% at the concentration of 10-6mmol/L of hIL-2-LuffinP1.The activity of inhibiting lymphocyte proliferation was proportional to the dosage.On the contrary,no obvious inhibition to lymphocyte proliferation was found in the control group treated with LuffinP1,the inhibition ratio only reached to 14% with the same concentration of hIL-2-LuffinP1.It was also found that,when hIL-2-LuffinP1 and LuffinP1 in different concentrations added to spleen cells stimulated by ConA,hIL-2-LuffinP1 still exhibited strong ability of inhibition on the splenocyte proliferation,while LuffinP1 has no obvious inhibiting effect on the lymphocyte proliferation in the two reaction systems mentioned above.MLR showed the same results.Furthermore,in the experiment of cytotoxic effects of IL-2-LuffinP1 on CTLL-2 cells,the inhibition rates of hIL-2-LuffinP1+IL-2 group and hIL-2-LuffinP1 group were 29.6% and 47.4% respectively,the difference was significant compared with IL-2 group(P

Objective: To study the inhibitory effect of retrovirus-mediated antisense human telomerase RNA (hTR) gene on hepatocelluar carcinoma, so as to explore an effective way to inhibit telomrerase activity in the treatment of hepatocellular carcinoma. Methods: Sense and antisense hTR gene were transfected into the packaging cell line PT67 by electroporation, and the stablely transfected cells were selected with G418. The recombinant retroviral supernatant was collected and transfected into HepG2 cells. After G418 selection, PCR was used to verify the integration of the hTR gene. Cell growth curves were drawn using MTT assay and the expression of PCNA was determined by immunofluorescence. TRAP-PCR-ELISA was adopted to detect the telomerase activity; cell cycle and apoptosis were evaluated by flow cytometry (FCM).Results: The expression of hTR gene could be amplified in HepG2-hTR-EcoRⅠ and HepG2-hTR-BamHⅠ cells, but not in untransfected HepG2 cells. The antisense hTR complementary to the template region of telomerase inhibited growth and proliferation of HepG2 cells. The expression of neutrophil proliferating cell nuclear antigen (PCNA) was decreased. Telomerase activity in the antisense hTR-treated group was (2.31?0.16),which was significantly lower than those of the other 2 groups(P

Objective To explore the effect of thyroid hormone on bone metabolism.Methods The thyroid in the rabbits had been cut off and the physics and histology of the bone in the rabbits were observed.The therapeutic effect of exterofection thyroid hormone was also observed.Results The lacking or reducing of thyroid hormone could lead to osteoporosis.The substitutive treatment of T_4 could partially inhibit the oesteopenia.Conclusion When the T_3 level equals to the level of T_4,the oesteopenia of the rabbits with thyroid being cut off may be caused by the lack of calcitonin.

Objective To probe the relationship between the expression of TL1A and the level of IFN-γ secreted by T cells in the acute stage of Guillain-Barre syndrome (GBS). Methods ① Six-week female Bal b/c mice were immunized by purified recombinant human soluble TNF-like molecular 1A (rhsTL1A) protein. The polyclonal antibody against rhsTL1A was identified by immunofluorescence using human umbilical vein epithelial cells (HUVEC). ② To detect the biologic activity of rhsTL1A, the peripheral blood mononuclear cells (PBMC) from the healthy donors were separated by Ficoll gradient centrifugation and were seeded on 96-well plates with medium containing 2 μg/ml PHA (control group), 2 μg/ml PHA + 25 ng/ml rhsTL1 A, 2 μg/ml PHA + 100 ng/ml rhsTL1A and 2 μg/ml PHA + 400 ng/ml rhsTLlA respectively. T cell proliferation assay was carried out using ~3H-TdR. ③ IFN-γ productions in the sera of the children with GBS in the acute stage were detected by ELISA. ④ The ratio of CD_3~+ TL1A~+ T cells to CD_3~+ T cells in the peripheral blood of the children with GBS in acute stage was detected with flow, cytometry. ⑤PBMC from the children in acute GBS were separated and cultured in the environment adding 2 μg/ml PHA and 400 ng/ml rhsTL1A in vitro. Then, the IFN-γ in the supernatant was determined by ELISA kit after 72 hours. Results ① hTL1A A expressed by eukaryotic HUVECs was recognized by rhsTL1 A polyclonal antiserum. ② The result of T cell proliferation assay showed that SI of 25 ng/ml rhTL1A, 100 ng/ml rhTL1A A and 400 ng/ml rhTL1A group was increased compared with control group. The SI of 2 μg/ml PHA +400 ng/ml rhsTL1 A group was the highest (2. 65) among them. ③ IFN-γ productions in the sera of the children with GBS in the acute stage ((102. 25±22. 17) pg/ml) were increased significantly compared with healthy control ((28.75 ± 1.31) pg/ml, t = 3. 309, P < 0. 05). ④ The ratio of CD_3~+ TL1A~+ T cells to CD_3~+ T cells in the peripheral blood of the children with GBS in acute stage (18.22%± 1.83%) was enhanced significantly compared with healthy control (5. 17% ±0. 48%, t = 6. 884, P < 0. 01). ⑤ PBMC both in healthy control and the acute GBS secreted more IFN-γ markedly ((43.56± 4.41) pg/ml and (180.64 ± 38.39) pg/ml) after being incubated in 2 μg/ml PHA and 400 ng/ml rhsTL1A (t =4. 523 and 2. 600, P <0. 01 and 0. 05 respectively). Moreover, PBMC in acute GBS secreted more IFN-γ, than that of the healthy group markedly (t = 3. 545, P < 0. 05). Conclusions ① The mouse antiserum recognizing rhsTL1A is successfully obtained. ② In this study, 400 ng/ml rhsTL1A promotes the proliferation of T cells activated by 2 μg/ml PHA, indicating that rhsTL1A has biological activity. ③ The expression of hTL1A of activated T cells in the peripheral blood of the children with acute GBS is up-regulated. These TL1A proteins promote the secretion of IFN-γ through binding to their receptors DR_3.

Objective:To investigate the correlation between circadian blood pressure pattern and heart rate variability and stroke severity and outcome in patients with acute ischemic stroke (AIS).Methods:Patients with first-ever AIS admitted to the Affiliated Qingdao Central Hospital of Qingdao University from January 2015 to January 2021 were retrospectively included. Ambulatory blood pressure monitoring (ABPM) and ambulatory electrocardiogram (AECG) were performed after admission. The severity of stroke was assessed according to the National Institutes of Health Stroke Scale. ≤8 were defined as minor stroke, and >8 were defined as moderate to severe stroke. The modified Rankin Scale was used to evaluate the clinical outcome at 3 months after onset. ≤ 2 were defined as good outcomes, and >2 were defined as poor outcomes. Multivariate logistic regression analysis was used to determine the independent influencing factors of stroke severity and outcome. Results:A total of 516 patients with AIS were enrolled, including 328 male (63.57%), aged 59.62±6.67 years old. Among them, 266 patients (51.55%) were in the minor stroke group and 250 (48.45%) were in the moderate to severe stroke group. There were 463 patients (89.73%) were in the good outcome group and 53 (10.27%) were in the poor outcome group. Multivariate logistic regression analysis showed that hypertension (odds ratio [ OR] 5.021, 95% confidence interval [ CI] 2.635-10.923; P<0.001), atrial fibrillation ( OR 3.896, 95% CI 2.574-8.521; P<0.001), circadian blood pressure pattern (non-dipper type: OR 2.436, 95% CI 1.031-4.749, P<0.001; reverse dipper type: OR 2.654, 95% CI 1.642-5.268, P<0.001), SDNN ( OR 0.298, 95% CI 0.114-0.730; P=0.002), SDANN ( OR 0.325, 95% CI 0.200-0.679; P=0.009), rMSSD ( OR 0.437, 95% CI 0.255-0.876; P=0.016) and pNN50 ( OR 0.369, 95% CI 0.291-0.767; P=0.013) were the independent influencing factors of stroke severity. Hypertension ( OR 4.857, 95% CI 1.957-8.552; P<0.001), baseline NIHSS score ( OR 2.189, 95% CI 1.597-3.315; P<0.001), stroke severity ( OR 3.853, 95% CI 2.316-5.958; P<0.001), circadian blood pressure pattern (non-dipper type: OR 2.997, 95% CI 1.128-5.430, P<0.001; reverse dipper type: OR 3.703, 95% CI 1.478-5.902; P<0.001), SDNN ( OR0.369, 95% CI 0.215-0.779; P=0.015), SDANN ( OR 0.372, 95% CI 0.198-0.862; P=0.018), rMSSD ( OR 0.455, 95% CI 0.314-0.896; P=0.026) and pNN50 ( OR 0.448, 95% CI 0.307-0.825; P=0.021) were the independent influencing factors of poor outcomes. Conclusion:The non-dipper and reverse dipper circadian blood pressure patterns and lower heart rate variability are independently associated with stroke severity and poor outcomes in patients with AIS.

Objective:To study the clinicopathological characteristics, treatment and prognosis in lupus nephritis (LN) patients with renal thrombotic microangiopathy (TMA), so as to provide more theoretical basis for clinicians to recognize and treat this disease.Methods:The clinical data of LN patients who underwent renal biopsy in the First Affiliated Hospital of Zhengzhou University from January 1, 2012 to May 31, 2019 were retrospectively collected and analyzed. According to renal clinicopathological examination, the patients were divided into renal TMA group and non-renal TMA group. The clinical data, laboratory examination, renal pathological examination, therapeutic measures and prognostic between the two groups were compared. Follow-up end points were defined as composite ends, including all-cause death, entry into end-stage renal disease, and estimated glomerular filtration rate decrease>50% of baseline. Kaplan-Meier survival curve and log-rank test were used to compare the difference of survival rate between the two groups, and multivariate Cox regression equation was used to analyze the risk factors of endpoint events in LN patients.Results:A total of 1 133 patients with LN were enrolled in this study. Patients with renal TMA were more likely to have hypertension ( χ2=16.310, P<0.001), higher baseline serum creatinine ( Z=-6.918, P<0.001) and 24-hour urine protein ( Z=-2.232, P=0.026), and higher renal pathology activity index (AI) score ( Z=1.957, P=0.001)and chronic index (CI) score ( Z=1.836, P=0.002). The proportions of hormone shock ( P<0.001) and plasma exchange ( P<0.001) in the renal TMA group were higher than those in non-renal TMA group. After treatment of (12±2) months, patients in the renal TMA group had a lower complete response rate ( χ2=10.455, P=0.001) and a higher non-response rate ( χ2=6.047, P=0.014) than those in non-renal TMA group, and were associated with worse prognosis (Log-rank test χ2=26.490, P<0.001). Renal TMA was an independent risk factor for poor prognosis ( HR=2.347, 95% CI 1.210-4.553, P=0.012). Conclusions:Compared with LN patients without renal TMA, LN patients with renal TMA are more likely to have hypertension, with higher serum creatinine, 24-hour urinary protein, AI and CI, suggesting poorer treatment response and renal prognosis. Moreover, renal TMA is an independent risk factor for poor prognosis in patients with LN.

Objective:To observe the inhibitory effects of Huangqi Jiedu Decoction on lung metastasis of breast cancer in nude mice; To explore the mechanism of intervening epithelial mesenchymal transformation (EMT) induced by Wnt/β-catenin signaling pathway.Methods:Totally 30 nude mice were divided into model group, adriamycin group and Huangqi Jiedu Decoction low-, medium-, and high-dosage groups according to random number table method. Each group was injected subcutaneously with mouse breast cancer 4T1 cells to construct tumor - bearing nude mice model. Huangqi Jiedu Decoction low-, medium- and high-dosage groups were intragastrically administrated with Huangqi Jiedu Decoction 17.82, 35.64 and 71.28 g/kg; adriamycin group was injected intraperitoneally adriamycin 0.05 g/kg; model group was intragastrically administrated with normal saline of the same volume for 21 d. Tumor volume was measured at 9, 15, and 21 days after modeling. After the end of administration, the tumor tissue was separated, the tumor weight was measured, and the tumor inhibition rate was calculated. The lung tissue was Isolated,, the number of lung metastatic nodules and the inhibition rate of lung metastasis was counted. HE staining was used to observe the tissue morphology and evaluate the effectiveness of the model. The protein expressions of β-catenin, E-Cadherin and Vimentin in lung tissue were detected by Western Blot. The mRNA levels of β-catenin, E-Cadherin and Vimentin in lung tissue were detected by real-time fluorescent quantitative PCR.Results:Compared with the model group, the tumor volume and mass of Huangqi Jiedu Decoction low-, medium- and high-dosage groups decreased ( P<0.01); the number of pulmonary metastasis nodules in Huangqi Jiedu Decoction high-dosage group significantly decreased ( P<0.01); the mRNA and protein expressions of β-catenin and Vimentinm decreased in the Huangqi Jiedu Decoction low-, medium- and high-dosage groups ( P<0.01), and the protein and mRNA expressions of E-Cadherin increased in the Huangqi Jiedu Decoction high-dosage group ( P<0.01). Conclusion:Huangqi Jiedu Decoction can effectively inhibit the growth and lung metastasis of breast cancer transplanted tumor, and the mechanism may be to down-regulate the expression of key molecules in the Wnt/β-catanin signaling pathway, thereby inhibiting the EMT process, so as to inhibit the lung metastasis of breast cancer.

Objective To investigate the dosimetric effect of same activity and same number of 125I seeds arranged in axial train but with different spacing.Methods A total of 27 film dosimeters were randomly and equally divided into group A,B and C.Each film was irradiated by three 125I seeds (activity of 1.48×107 Bq).The seeds were arranged in line,and their axial spacing was 1 mm,5 mm and 10 mm respectively.Image analysis software was used to draw iso-gray contour curves of 20,25,30 and 40 gray value on the films,and to calculate the areas contained by each curve.Results Multi-sample mean comparison variance analysis showed that the differences in area contained by 20 and 25 iso-gray contour curves were statistically significant between each other among the three groups (P＜0.001).The difference in area contained by 30 iso-gray contour curve between group A and group B was not statistically significant (P＞ 0.05),while the difference in area contained by 30 iso-gray contour curve between group A and group C as well as between group B and group C was statistically significant (P＜0.001).No statistically significant difference in area contained by 40 iso-gray contour curve existed between each other among the three groups (P=0.99).Conclusion Different spacing arrangement of same activity and same number of 125I seeds can directly influence the peripheral dose distribution.In specific dose range,seed-spacing of 1 mm arrangement may obtain better dose distribution than seed-spacing of 5 mm or 10 mm arrangement can do.

ObjectiveTo investigate the mechanism of Chaihu Qinggantang （CHQGT） in the treatment of granulomatous lobular mastitis （GLM） in the rat model. MethodSixty female rats were divided into a normal group， a model group， a prednisolone group （0.001 8 g·kg^-1）， and three CHQGT low-dose， medium-dose， and high-dose groups （4.5， 8.9， 17.8 g·kg^-1）. The tissue homogenates mixed with GLM lesion tissue and Fritner's reagent were used for modeling. After modeling， the treatment groups were given corresponding treatment factors， and the normal group and the model group were given the equal volume of normal saline. The changes in mammary gland of rats were observed after 14 d. Hematoxylin-eosin （HE） staining was used to observe the histopathological changes in breast samples. The mRNA expressions of NOD-like receptor thermal protein domain associated protein 3 （NLRP3） inflammasome， Caspase-1， and interleukin-1β （IL-1β） were detected by real-time quantitative fluorescence polymerase chain reaction （Real-time PCR）. The protein expressions of NLRP3， Caspase-1， IL-1β， and IL-18 were detected by Western bolt. ResultAs compared with the normal group， the breasts of rats in the model group were obviously swelling， and mammary gland inflammation index was significantly increased （P<0.01）. Pathological changes included the formation of granuloma centered on the lobule of mammary gland with a large number of inflammatory cells such as lymphocytes and plasma cells. The mRNA expressions of NLRP3， Caspase-1， and IL-1β， and the protein expressions of NLRP3， Caspase-1， IL-1β， and IL18 in the model group were significantly increased （P<0.01）. Compared with the model group， the treatment groups improved breast swelling， and the CHQGT medium and high-dose groups and the prednisolone group reduced inflammation index to some extent after treatment （P<0.05， P<0.01）. The inflammation degree of mammary gland was significantly improved， and inflammatory cells such as macrophages， lymphocytes， and plasma cells were reduced to varying degrees in pathological aspects. The mRNA expressions of NLRP3， Caspase-1， and IL-1β， and the protein expressions of NLRP3， Caspase-1， IL-1β， and IL-18 in the CHQGT high-dose group and the prednisolone group were significantly down-regulated （P<0.05， P<0.01）. ConclusionCHQGT inhibits inflammation and treats GLM in rats. The mechanism is possibly related to the inhibition of NLRP3/IL-1β signaling pathway， which provides a new target for the prevention and treatment of GLM by Qingxiao method.

Objective: Investigate osteogenic differentiation of canine periodontal ligament stem cells ( cPDLSCs) via over-expression ephrinB2 in cPDLSCs． Methods : cPDLSCs were isolated from the premolars and molars of Beagle．After transfected with EfnB2-GFP-Bsd and GFP-Bsd empty Vector，cPDLSCs were induced to osteogenic differentiation．Western blot was used to invest the expression of ephrinB2 protein．The effect of osteogenic differentiation of EfnB2-cPDLSCs and Vector-cPDLSCs were analyzed by RT-PCR , CCK-8，Alizarin-red S staining and ALP. Results: There was no significant difference in cell proliferation between EfnB2-cPDLSCs and Vector-cPDLSCs．While EfnB2-cPDLSCs displayed an enhanced ALP activity and more prominent mineralized nodules compared with Vector-cPDLSCs．The odonto-/ osteogenic genes in EfnB2-cPDLSCs were also highly enhanced． Conclusion The results of our study indicated that ephrinB2 gene-transfected cPDLSCs showed enhanced osteogenic differentiation.