Objective: To constructCSF1R+/-mice and to analyze their genotypes, so as to provide animal model basis for disease pathological mechanism and drug target. Methods : A linearized targeting vector was designed according to Cre/Loxp system. A Loxp site was inserted upstream of the 5th exon of theCSF1Rgene, and a neomycin resistance box with Loxp sites on both sides was inserted downstream of the 5th exon. The linearized targeting vector was electroporated into embryonic stem cells. The correctly targeted embryonic stem cells were injected into the blastocysts of C57BL/6J mice to obtain chimeric mice, which were bred with Zp3-Cre mice. The newborn mice were numbered 9-14 days after birth and their tails were cut. The DNA of the mice was extracted, and the genotype of the mice was identified by polymerase chain reaction and agarose gel electrophoresis. The expression of CSF1R in mouse macrophages was detected by flow cytometry. The expression of CSF1R in mouse tissues was detected by Western blot. Results: The results of agarose gel electrophoresis showed that 453 bp bands were amplified in wild type mice, and 453 bp and 650 bp bands were amplified in heterozygous mice. The results of flow cytometry showed that the expression of CSF1R in peritoneal macrophages and bone marrow-derived macrophages of CSF1R heterozygous mice was lower than that of WT group(P<0.05). The results of Western blot showed that the expression of CSF1R in spleen, kidney and brain tissue of CSF1R heterozygous group was lower than that of WT group(P<0.05). Conclusion CSF1R+/-mice are successfully constructed, reproduced and identified, which provides an animal model basis for further revealing the potential mechanism of CSF1R in immune regulation.

Objective: To construct microRNA(miR)-22 gene knockout(miR-22-/-) mice using CRISPR/Cas 9 technology, to breed miR-22-/- mice and to identify their genotypes. Methods : In this experiment, CRISPR/Cas 9 technology was used to construct miR-22-/- genetically engineered mice. After gene identification, the F0 generation miR-22-/- mice were mated with wild-type mice in the same litter to obtain F1 generation miR-22-/- mice. The miR-22 knockout efficiency was analyzed at the RNA level by real-time fluorescence quantitative polymerase chain reaction(qPCR). Western blot was used to detect the interaction between miR-22 and target genes. Results : miR-22-/- mice were successfully constructed using CRISPR/Cas 9 technology, gene identification was performed on the bred mice, and three stable genotypes of miR-22+/+,miR-22+/-, and miR-22-/- were identified. The real-time fluorescence quantitative PCR detection results confirmed that miR-22-/- mice showed almost no expression of miR-22 in the heart, liver, lung, kidney, spleen, and thymus tissues compared to wild-type mice in the same litter. Western blot analysis showed that the relative expression level of NLRP3 protein in miR-22-/- mouse tissues was lower than that in wild-type mice. Conclusion A miR-22-/- mouse model is successfully constructed, and stable genetic homozygous miR-22-/- mice is obtained. This indicates that miR-22 has an inhibitory effect on the downstream target gene NLRP3.

Objective: To explore the breeding and genotyping of CCR2 knockout mice, and to verify the applicability of the polymerase chain reaction(PCR) method for genotype detection of CCR2 knockout mice. Methods: The introduced CCR2 pure male mice and wild-type female mice were mated and bred to produce the offspring generation, the obtained F1 generation heterozygous mice were continued to be mated. DNA was extracted by clipping the tail tissues of the mice at the age of 2 weeks, the target gene fragment was amplified by PCR, and the genotypic results were determined by agarose gel electrophoresis. The proportion of purebred progeny carrying the CCR2 knockout gene was increased by genetic crosses, the effect of CCR2 knockout in the progeny mice was verified by using Western blot against major immune cells and key organs, and flow cytometry was used to detect whether the knockout of the CCR2 gene had any effect on the function of the immune system by targeting the major immune cells. Results: CCR2 knockout mice were successfully bred and characterized, and three genotypes of F2 generation mice were obtained: CCR2+/+, CCR2+/-, and CCR2-/-. The offspring genotypes were identified by PCR, and Western blot showed extremely low CCR2 protein expression in CCR2 knockout mice. Flow analysis showed that CCR2 knockdown reduced the expression of CD4+T and Th1 cells in mouse spleen-derived T cells, but did not affect macrophage function. Conclusion Correct breeding and identification are important ways to get the pure CCR2 knockout mice, and PCR method for identifying mouse genotypes is simple, fast and reliable.

Objective To construct myeloid-specific Spi1 gene knockout mice and analyze their genotypes,so as to provide animal model basis for the study of pathological mechanism of diseases and drug targets.Methods Ac-cording to the principle of CRISPR/Cas9 technology and Cre/LoxP system,sgRNA and Donor vectors were de-signed and constructed.The transcript of Exon 2(Exon 2)was used as the knockout region,and Loxp elements were placed on both sides of Exon 2.Cas9 protein,sgRNA and Donor vector were mixed and microinjected into the fertilized eggs of C57BL/6J mice,the fertilized eggs were transplanted into the uterus of C57BL/6J pregnant female mice,and F0 generation was obtained after 19～20 days.Positive F0 mice were mated with C57BL/6J mice to ob-tain stable F1 Spi1flox/+mice.Spi1flox/+mice of F1 generation were selfed to obtain Spi1flox/flox mice.Spi1flox/flox mated with Lyz2-Cre+mice to obtain Spi1flox/+/Lyz2-Cre+mice,and then mated with Spi1flox/flox,the Spi1flox/flox/Lyz2-Cre+mice were myeloid-specific Spi1 gene knockout(KO)mice.Spi1flox/flox/Lyz2-cre-mice were used as wild-type(WT)mice.DNA of WT and KO mice was extracted,and the genotypes were identified by agarose gel electro-phoresis after PCR amplification.Western blot was used to detect the expression of spleen focus forming virus provi-ral integration oncogene,Spi-1/purine rich box-1(PU.1)in immune cells of WT and KO mice.Results The results of PCR identification showed that the genotype of mice with only 220 bp amplified by flox primer was Spi1flox/flox homozygote,and the genotype of mice with 700 bp amplified by Lyz2-Cre primer was Lyz2-Cre+.Western blot showed that compared with WT group,the protein PU.1 was not expressed in bone marrow-derived macropha-ges(BMDMs)and peritoneal macrophages(PM)in KO group(P<0.01).There was no significant difference of statistics in the expression level of PU.1 in T cells between KO mice and WT mice.The results of PCR and West-ern blot showed that myeloid-specific Spi1 KO mice were successfully constructed.Conclusion The myeloid-spe-cific Spi1 gene KO mice are successfully constructed and identified,which provides animal model basis for further revealing the potential mechanism of PU.1 inimmune regulation.

Objective To breed and identify the T lymphocyte-conditional Spi1 knockout mice for the further in-vestgation of the specific role of Spi1-encoded protein PU.1.Methods The Lck-Cre mice were mated with Spi1flox/flox mice to obtain Lck-Cre×Spi1flox/flox mice(T lymphocyte-specific Spi1 knockout mice),and the genotype was determined by polymerase chain reaction(PCR)and agarose gel electrophoresis.Magnetic beads were used to sort out the splenic T lymphocytes,and the knockdown efficiency of PU.1 in T cells was detected by Western blot,quantitative real-time PCR(qPCR)and flow cytometry.Results The Lck-Cre×Spi1flox/flox mouse genotype was stably inherited.Compared with Spi1flox/flox mice,the expression level of PU.1 was significantly reduced in splenic T cells of Lck-Cre×Spi1flox/flox mice.Conclusion In this study,the T lymphocyte-specific Spi1 knockout mice was successfully constructed by applying Cre/LoxP system and CRISPR/Cas9 technology,which provided a reliable an-imal model for the subsequent experiments of the specific role of PU.1 in T cell-related diseases.

Objective To construct Csf1r-CreERT2 R26REYFP reporter gene mice and assess the efficacy of Csf1r-CreERT2-mediated enhancement of CSF1R in CD45+cells labeled with yellow fluorescein protein EYFP.Methods Csf1r-CreERT2 mice were crossbred with R26REYFP homozygous mice,and Csf1r-CreERT2R26REYFP mice were identified through PCR and Western Blot analyses.Flow cytometry was employed to evaluate CSF1R tag-efficiency in CD45+cells across different mouse tissues following tamoxifen induction.Results Csf1r-CreERT2 R26REYFP reporter gene mice were acquired.In addition,it was found that Csf1r-CreERT2-mediated EYFP could effectively mark CSF1R in various tissues of mice and CD45+cells in different locations.Compared to the R26REYF P group,the highest labeling efficiency was observed in the brain tissue(P＜0.001),the lowest in the thymus tissue(P＜0.05),and no sig-nificant difference was observed in the spleen tissue.Conclusion Adult Csf1r-CreERT2 mice and R26REYFP mice are effective ways to obtain Csf1r-CreERT2 R26REYFP induced conditional fluorescence mice.Csf1r-CreERT2 can mediate EYFP to effectively trace CSF1R in CD45+cells in different parts of mice.

Objective:To investigate the clinical effect of dapagliflozin combined with metformin on type 2 diabetes mellitus (T2DM).Methods:A total of 100 patients with T2DM who received treatment in The Second People's Hospital of Hefei from June 2019 to May 2021 were included in this study. They were randomly divided into a control group ( n = 50) and an experimental group ( n = 50). The control group was treated with metformin, and the experimental group was treated with dagglitazin combined with metformin. All patients were treated for 3 months. Blood glucose index, blood lipid level, and the incidence of adverse reactions were compared between the two groups. Results:After treatment, fasting blood glucose, 2-hour post-prandial blood glucose, and glycosylated hemoglobin in the experimental group were (5.56 ± 0.37) mmol/L, (8.32 ± 0.23) mmol/L, and (6.17 ± 0.26)% respectively, which were significantly lower than (6.96 ± 0.48) mmol/L, (9.58 ± 0.39) mmol/L, and (7.27 ± 0.26)% respectively in the control group ( t = 3.59, 6.92, 5.03, all P < 0.05). The total cholesterol and triglyceride in the experimental group were (3.58 ± 0.53) mmol/L and (1.25±0.26) mmol/L, respectively, which were significantly lower than (4.94 ± 0.58) mmol/L and (1.93 ± 0.18) mmol/L in the control group ( t = 3.16, 4.25, both P < 0.05). There was no significant difference in the incidence of adverse reactions between the two groups ( P > 0.05). Conclusion:Dapagliflozin combined with metformin can effectively control blood glucose and blood lipid in T2DM patients without increasing adverse reactions.

Chronic obstructive pulmonary disease (COPD) has a high global morbidity and mortality and a severe disease burden, yet progress in treatment and prevention has been slow in recent decades. Early COPD has few symptoms and is severely underdiagnosed and undertreated; it is crucial to search for effective clues of early COPD and provide management interventions. By reviewing the definition, risk factors, diagnosis and management interventions, this study explores the disease evolution of early-stage COPD, which can help clinical practice to develop more effective preventive and therapeutic strategies for stopping or slowing down the natural progression of the disease, improving the long-term prognosis, and reducing the disease burden.
Humans ; Pulmonary Disease, Chronic Obstructive/drug therapy* ; Prognosis ; Risk Factors

Objective:To evaluate the application value of patient-based real-time quality control (PBRTQC) algorithms in intralaboratory comparison between various hematology analyzers.Method:From April 1 st 2020 to March 31 th 2021, data of white blood cell (WBC) counts and daily comparison results of fresh venous blood, measured by five hematology analyzers, were collected at the Department of Laboratory Medicine in Hebei Children′s Hospital. First, the professional intelligent PBRTQC software system was applied to conduct the parameter setting, program establishment, and performance verification. Three concentration ranges of WBC were selected, low concentration (2.5-4.5)×10 9/L, medium concentration (6.0-8.0)×10 9/L and high concentration (12.0-14.0)×10 9/L for the comparison. Next, WBC counts were calculated with both of the EWMA and median methods, the results were then analyzed by PBRTQC using the module of"intralaboratory comparison of hematology analyzers". Finally, bias of intralaboratory comparison among various hematology analyzers analyzed by means of EWMA and daily comparison results of fresh venous blood were compared. Based on the standard of WS/T 406-2012,allowable error ±7.50% in WBC counts was set as the relative bias standard among different instruments. Results:(1) A total of 38 313 sample results were included, there were 70 warning results out of these samples based on the EWMA quality control method established on the data of patients with white blood cell count in our laboratory, with an early warning rate of 0.183‰, a probability of error detection of 100%, and a probability of false loss of control of 0. EWMA quality control efficiency met the quality objectives. (2) In the comparison monitoring of the results of 5 blood cell analyzers at high concentrations, the coincidence rate between EWMA and median method were both 100% (46/46) in weekly and monthly comparison, and EWMA could maintain a relatively stable monitoring efficiency in daily comparison. (3) In the selected natural month, the consistency rate between EWMA method and fresh blood comparison method was 95.24% (20/21).Conclusion:PBRTQC can be used as a valuable supplementary tool of IQC to continuously and effectively monitor the consistency of data derived from intralaboratory hematology analyzers with different bands and types, which can not only reduce the risk of quality and operating costs, but also improve the efficiency of laboratory management.

Objective:Chemiluminescence immunoassay was used to detect the levels of anticardiolipin antibody (aCL) -IgA/IgG/IgM and anti-β2-glycoprotein Ⅰ antibody (aβ2GPⅠ) -IgA/IgG/IgM in healthy non-pregnant and pregnant women to explore the changes of antiphospholipid antibody in different pregnancy periods.Methods:This prospective study was conducted in Shandong Provincial Maternal and Child Health Care Hospital Affiliated to Qingdao University, involving normal pregnant women who underwent prenatal examination and healthy non-pregnant women with no history of adverse pregnancy who underwent progestational eugenic health examination from April 2020 to August 2021. The levels of aCL-IgA/IgG/IgM and aβ2GPⅠ-IgA/IgG/IgM were detected using BIO-FLASH chemiluminescence immunoassay analyzer and P95 as well as P99 were calculated, respectively. The difference in the six data between non-pregnant and pregnant women was compared using Mann-Whitney U test. Kruskal-Wallis H test was used to compare the change of each antibody in different pregnancy periods and Spearman correlation was used to analyze the correlation between different trimester and the levels of aCL-IgA/IgG/IgM and aβ2GPⅠ-IgA/IgG/IgM. Results:A total of 454 cases met the inclusion criteria, and 435 cases were included in the analysis after excluding 19 cases, among them 110 were non-pregnant women and 325 were pregnant women, including 110 cases in the first trimester (≤13 +6 weeks), 110 cases in the second trimester(14 +0-27 +6 weeks), and 105 cases in the third trimester (≥28 weeks). P99 value of aCL-IgA/IgG/IgM and aβ2GPⅠ-IgA/IgG/IgM in the non-pregnant women were 7.31, 14.70, 7.92, 3.58, 13.60, and 4.95 CU, which in the pregnant women were 5.90, 12.78, 5.70, 1.60, 10.65, and 3.90 CU, and were all lower than the cut-off value of 20 CU that given by the analyzer manufacturer. The levels of aCL-IgA/IgG/IgM, and aβ2GPⅠ-IgG/IgM in the pregnant women were significantly decreased comparing with the non-pregnant women [aCL-IgA: 1.90 CU (1.40-2.70 CU) vs 2.90 CU (2.20-3.83 CU), Z=－7.14; aCL-IgG: 3.00 CU (2.20-4.50 CU) vs 6.10 CU (4.20-7.83 CU), Z=－10.26; aCL-IgM: 1.40 CU (1.10-2.30 CU) vs 2.65 CU (2.08-3.73 CU), Z=－8.87; aβ2GPⅠ-IgG: 3.50 CU (2.60-4.90 CU) vs 4.75 CU (3.60-5.93 CU), Z=－5.45; aβ2GPⅠ-IgM: 0.70 CU (0.50-1.20 CU) vs 1.00 CU (0.60-1.53 CU) , Z=－3.73; all P<0.001]. The aCL-IgA level in the third trimester was higher than those in the first and second trimester (both P<0.05). The levels of aCL-IgG/IgM in the second trimester and aβ2GPⅠ- IgG in the second and third trimesters were significantly decreased than those in the first trimester (all P<0.05). Spearman analysis showed that aCL-IgG/IgM, aβ2GPⅠ-IgA/IgM had no significant correlation with the pregnancy period (the first, second and the third trimester) (all P>0.05). However, a weak correlation between the aCL-IgA, aβ2GPⅠ- IgG and the pregnancy period was observed ( r=0.28 and－0.49, both P<0.001) Conclusions:P99 value of aCL-IgA/IgG/IgM and aβ2GPⅠ-IgA/IgG/IgM levels in normal pregnant women and non-pregnant women are lower than the cut-off value of 20 CU given by the analyzer manufacturer. The levels of aCL-IgA/IgG/IgM and aβ2GPⅠ-IgG/IgM during pregnancy are lower than those before pregnancy and fluctuate with the pregnancy period, but have no significant correlation with the pregnancy period. The clinical diagnosis of antiphospholipid syndrome should be made according to the cut-off values of aCL-IgA/IgG/IgM and aβ2GPⅠ-IgA/IgG/IgM determined by each laboratory.