1.Mechanism of Bushen Kaixuan Tongluo Prescription in Improving Diabetic Nephropathy Based on cAMP Signaling Pathway
Miao XU ; Baosheng ZHAO ; You WANG ; Yuzhuo CHANG ; Zehao LIU ; Lingling QIN ; Haiyan WANG ; Ming GAO ; Cuiyan LYU ; Tonghua LIU
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(11):87-96
ObjectiveTo investigate the molecular mechanism by which the Bushen Kaixuan Tongluo prescription exerts a renal protective effect in mice with diabetic kidney disease (DKD) by regulating the cyclic adenosine monophosphate (cAMP) signaling pathway. MethodsThirty specific pathogen-free (SPF) male db/db mice were adaptively fed for three weeks. Mice with a random tail vein blood glucose level ≥ 11.1 mmol·L-1 and urinary albumin-creatinine ratio (ACR) ≥ 30 mg·g-1 were considered successfully modeled. The successfully modeled mice were randomly divided into five groups with six mice in each group: the model group, the low-, medium-, and high-dose Bushen Kaixuan Tongluo prescription groups (administered at doses of 7, 14, 28 g·kg-1·d-1 respectively), and the positive drug irbesartan group (administered at a dose of 20 mg·kg-1·d-1). Additionally, six db/m mice were selected as the blank group. Mice in each group were given intragastric administration of the Bushen Kaixuan Tongluo prescription at the corresponding concentrations, irbesartan, or an equal volume of pure water, and the intervention lasted for 12 weeks. During the experiment, the general conditions, body weight changes, and renal function indicators of the mice were dynamically monitored. After the intervention, a blood glucose meter was used to measure the fasting blood glucose (FBG) of the mice. An automatic biochemical analyzer was employed to detect the levels of serum creatinine (SCr), blood urea nitrogen (BUN), urinary microalbumin (uALB), ACR, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC), triglycerides (TG), leptin (LEP), glycosylated serum protein (GSP), and insulin (INS) in the mice. Renal tissues were collected for hematoxylin-eosin (HE) staining, periodic acid-Schiff (PAS) staining, and Masson's trichrome staining to observe the histopathological changes. Immunohistochemistry (IHC) was used to detect the expressions of protein kinase A (PKA) and cAMP response element-binding protein (CREB) in the mice. Western blot analysis was performed to determine the expression levels of PKA, phosphorylated protein kinase A (p-PKA), CREB, phosphorylated cAMP response element-binding protein (p-CREB), and B-cell lymphoma-2 (Bcl-2) proteins in the renal tissues of the mice. Real-time quantitative polymerase chain reaction (Real-time PCR) was used to detect the mRNA expression levels of PKA, CREB, and Bcl-2 in the renal tissues of the mice. ResultsCompared with the blank group, the mice in the model group showed listlessness, decreased activity, and a significant increase in body weight (P<0.01). Biochemical indicators revealed that the levels of BUN, uALB, ACR, AST, ALT, TC, TG, FBG, LEP, GSP, and INS were significantly increased (P<0.01), while SCr showed an increasing trend with no statistically significant difference. Compared with the model group, the mice in the Bushen Kaixuan Tongluo prescription intervention groups had improved general conditions and a decreasing trend in body weight. Biochemical indicators showed that the levels of BUN, uALB, ACR, TC, GSP, and INS were significantly decreased (P<0.05), while SCr, AST, ALT, TG, and LEP showed a decreasing trend with no statistically significant difference. Renal histopathological analysis showed that the model group exhibited typical DKD pathological features such as thickening of the glomerular basement membrane, expansion of the mesangial matrix, and deposition of collagen fibers in the renal tubulointerstitium, and all treatment groups could alleviate the above pathological damages. The IHC results showed that compared with the blank group, the expression levels of p-PKA and p-CREB in the renal tissues of the model group were significantly decreased (P<0.01). Compared with the model group, the expression level of p-PKA in the medium-dose Bushen Kaixuan Tongluo prescription group was significantly increased (P<0.01), while the expression level of p-CREB showed an increasing trend with no statistically significant difference. Western blot results showed that compared with the blank group, the expression levels of p-PKA/PKA, p-CREB/CREB, and Bcl-2 in the model group were significantly decreased (P<0.05). Compared with the model group, the expression levels of these proteins in the medium-dose Bushen Kaixuan Tongluo prescription group were significantly increased (P<0.01). Real-time PCR results showed that compared with the blank group, the mRNA expressions of PKA, CREB, and Bcl-2 in the model group were significantly down-regulated (P<0.05). Compared with the model group, the mRNA expressions of these genes in the medium-dose Bushen Kaixuan Tongluo prescription group were significantly up-regulated (P<0.05). ConclusionThe Bushen Kaixuan Tongluo prescription can improve the liver and kidney functions of db/db mice, correct lipid metabolism disorders and glucose metabolism imbalance. Its renal protective effect is associated with up-regulating the cAMP signaling pathway to improve renal fibrosis and reduce the level of oxidative stress, thereby protecting renal function.
2.Effect of functional pelvic floor muscle training on stress urinary incontinence in women
Ya'nan LI ; Miao YE ; Juan WU ; Cong CHEN ; Lingfang WAN ; Lili YU ; Linlin GAO ; Yi QIN ; Huafang JING
Chinese Journal of Rehabilitation Theory and Practice 2026;32(6):690-698
ObjectiveTo observe the effect of functional pelvic floor muscle training on pelvic floor muscle function, incontinence symptoms and quality of life in female patients with stress urinary incontinence (SUI). MethodsFrom February, 2024 to July, 2025, 40 female patients with SUI were recruited from Beijing Bo'ai Hospital and advertisements. They were randomly divided into control group (n = 20) and experimental group (n = 20). The control group received conventional pelvic floor muscle contraction and relaxation training, and the experimental group received functional pelvic floor muscle contraction and relaxation training, for eight weeks. The data of pelvic floor muscle strength, pelvic floor muscle surface electromyography (sEMG), 1-hour urine pad test and Incontinence Quality of Life questionnaire (I-QOL) were collected before and after training. The compliance rate was counted after training. ResultsOne case dropped down in the control group and three cases dropped down in the experimental group. After treatment, the pelvic floor muscle strength increased in both groups (|Z| > 3.317, P < 0.01), the pre-resting average value, sustained contraction average value and durable contraction average value of sEMG improved in the control group (|t| > 2.731, P < 0.05), the mass of 1-hour urine pad significantly reduced (t > 9.215, P < 0.001) and the I-QOL score significantly increased (|t| > 13.229, P < 0.001) in both groups; compared with the control group, the pelvic floor muscle strength significantly increased (Z = -2.281, P = 0.023), the mass of 1-hour urine pad significantly reduced (t = 4.215, P < 0.001), the I-QOL score significantly increased (t = -4.501, P < 0.001), and the compliance rate significantly increased (Z = -2.798, P < 0.01) in the experimental group . ConclusionFunctional pelvic floor muscle training can significantly improve pelvic floor muscle strength, incontinence symptoms and quality of life in female patients with SUI.
3.Bibliometric analysis of hot topics and trends in thyroid cancer nursing research at home and abroad from 2004 to 2023
Yu JIANG ; Shiyu YIN ; Chunyuan QIN ; Junping ZHONG ; Miao XIANG ; Zhaoting CHENG
Chinese Journal of Practical Nursing 2025;41(25):1970-1977
Objective:To analyze the hotspots and trends in thyroid cancer nursing research from 2004 to 2023 at home and abroad through bibliometric methods, so as to provide references and ideas for future research in this field.Methods:A systematic search was conducted for core journal documents on thyroid cancer nursing research included in China National Knowledge Infrastructure, Wanfang Database, VIP Chinese Journal Network and Web of Science Core Collection from January 1, 2004 to December 31, 2023, using CiteSpace6. 3.R1 software performs visual analysis.Results:A total of 119 domestic documents and 417 international documents were included for analysis. From 2004 to 2023, the number of publications in the field of thyroid cancer nursing research at home and abroad showed an upward trend. Domestic research hotspots mainly include four directions: perioperative nursing, symptom nursing, care for radioactive iodine-131 treatment, and physical and mental health promotion. Foreign research hotspots mainly include three directions: nursing practice exploration and guideline formulation, care for radioactive iodine-131 treatment, and quality of life.Conclusions:The field of thyroid cancer nursing has received continuous attention from scholars at home and abroad. In future research, it is recommended that domestic scholars actively carry out cross-institutional and multi-center team cooperation in accordance with local needs, focus on the research of physical and mental care and health promotion of thyroid cancer patients at different survival stages, and the construction of clinical nursing guidelines. With the help of core research institutions and expert teams, actively promote the development of high-quality nursing research in this field.
4.Epidemiological characteristics and treatment outcome of pulmonary tu-berculosis patients combined with diabetes mellitus in Bijie City from 2017 to 2022
Mai ZHANG ; Jingyuan YANG ; Qin YU ; Miao YU ; Jinlan LI ; Yong LIU
Chinese Journal of Infection Control 2025;24(3):402-409
Objective To analyze the epidemiological distribution characteristics and influencing factors of treat-ment outcome of pulmonary tuberculosis(PTB)patients complicated with diabetes mellitus(DM)in Bijie City from 2017 to 2022,and provide reference for formulating prevention and control measures of PTB-DM.Methods The registered management cases of PTB patients combined with DM from the tuberculosis surveillance report informa-tion management system in Bijie City in 2017-2022 were collected.Changing trends in annual registration rates and successful treatment rates were analyzed with Joint-point regression models.Different characteristics between pa-tients with PTB-DM and PTB alone were conducted comparative analysis;the influencing factors of treatment out-come were analyzed by x2 test and binary logistic regression analysis.Results A total of 679 PTB-DM patients were registered in Bijie City from 2017 to 2022,accounting for 1.53%of total PTB patients,the annual registration rate increased from 1.11/100 000 in 2017 to 3.08/100 000 in 2022,with an increasing trend in the annual percentage change(APC=26.67%,95%CI:5.36%-52.29%,t=3.564,P=0.024).In the analysis on characteristics,the proportion of PTB-DM male patients,age ≥45 years and farmers,the ratio of direct consultation to transfer treat-ment,the pathogenicity positive rate,the proportion re-treatment,and rate of delay in consultation were all higher than those of PTB alone;successful treatment rate and proportion of floating population were all lower than PTB alone,difference were all statistically significant(all P<0.05).The successful treatment rate of PTB-DM patients in Bijie City from 2017 to 2022 was 87.80%.Age,occupation,pathogenicity diagnosis results were influencing fac-tors for treatment outcome in patients with PTB-DM,with farmers(OR=3.68,95%CI:1.22-11.09),with pathogenicity positivity(OR=2.84,95%CI:1.24-6.50),and without pathogenic detection result+tuberculous pleurisy(OR=11.35,95%CI:2.16-59.74)being risk factors for successful treatment.Conclusion Although the proportion of PTB-DM in the total PTB patients in Bijie City from 2017 to 2022 was not high,it showed an up-ward trend;it is necessary to attach great importance to male,re-treated,farmer,and pathogenicity positivity co-morbid patients in Bijie City,and strengthen the two-way screening of PTB-DM in high-risk populations.
5.Effect of LncRNA CTBP1-AS2 on malignant biological behavior of acute myeloid leukemia cells by regulating miR-433-3p/DPP8 signaling axis
Suhui LIU ; Lijuan DUAN ; Chao LI ; Fan QIN ; Miao SHANG
Chinese Journal of Immunology 2025;41(11):2631-2636
Objective:To investigate effect of LncRNA C-terminal binding protein 1 antisense RNA 2(CTBP1-AS2)on malig-nant biological behavior of acute myeloid leukemia(AML)cells by regulating miR-433-3p/dipeptidyl peptidase 8(DPP8)signaling axis.Methods:AML cells HL-60 were randomly separated into si-NC group,si-CTBP1-AS2 group,si-CTBP1-AS2+anti-NC group,and si-CTBP1-AS2+anti-miR-433-3p group.CCK-8 method and EdU staining were applied to detect HL-60 cell proliferation.Flow cytometry was applied to detect HL-60 cells apoptosis.Transwell was applied to detect migration and invasion of HL-60 cells.Western blot was applied to detect expressions of PCNA,Bax,MMP-9 and DPP8 proteins in HL-60 cells.ELISA was applied to detect expres-sions of IFN-γ and IL-1β.Dual luciferase reporter gene experiment was applied to analyze interaction between LncRNA CTBP1-AS2 and miR-433-3p,and between miR-433-3p and DPP8.Results:Expressions of LncRNA CTBP1-AS2,DPP8 mRNA,A450,EdU posi-tive cells,number of migrating cells,number of invading cells,PCNA protein,MMP-9 protein,DPP8 protein and IL-1β protein ex-pressions of HL-60 cells in si-CTBP1-AS2 group were lower than si-NC group,miR-433-3p expression,apoptosis rate,Bax protein and IFN-γ protein expressions were higher than si-NC group(P<0.05).Compared with si-CTBP1-AS2 group and si-CTBP1-AS2+anti-NC group,miR-433-3p expression,apoptosis rate,and expressions of Bax protein and IFN-γ protein in si-CTBP1-AS2+anti-miR-433-3p group were decreased,DPP8 mRNA,A450,EdU positive cells,number of migrating cells,number of invading cells,PCNA protein,MMP-9 protein,DPP8 protein and IL-1β protein expressions were increased(P<0.05).LncRNA CTBP1-AS2 targeted negative regula-tion of miR-433-3p,while miR-433-3p targeted negative regulation of DPP8.Conclusion:Knocking down LncRNA CTBP1-AS2 may inhibit malignant biological behavior of AML cells,whose mechanism may be achieved by regulating miR-433-3p/DPP8 signaling axis.
6.Application effects of learning pyramid theory combined with constructivism in the standardized training of gastroenterology nurses
Miao CHEN ; Qin MENG ; Xiuping LIU ; Ying WANG ; Xiaoyu CHEN ; Gui JIA
Chinese Journal of Medical Education Research 2025;24(5):686-690
Objective:To investigate the application effects of learning pyramid theory combined with constructivism in the standardized training of nurses in the department of gastroenterology.Methods:We randomly divided 104 gastroenterology nurses who received standardized training from October 2021 to October 2023 into observation group and control group, with 52 nurses in each group. The control group received conventional teaching, while the observation group received a combination of learning pyramid theory and constructivist teaching. The two groups were compared in terms of theoretical and practical scores, core competencies, self-learning abilities, and professional values scores before and after training. SPSS 23.0 was used to perform the t-test. Results:After training, the two groups showed improvements in all the tests; and the scores were all significantly higher in the observation group than in the control group: theoretical knowledge [(88.61±15.62) vs. (81.76±14.27)], skill practice [(82.91±15.17) vs. (76.40±13.82)], Competency Inventory for Registered Nurses scale score [(157.83±21.73) vs. (125.40±15.77)], self-learning ability score [(123.38±21.62) vs. (97.43±15.23)], and Nurse Professional Values scale score [(89.15±14.63) vs. (78.52±12.84)].Conclusions:The teaching model of learning pyramid theory and constructivism theory can enhance standardized training effectiveness for gastroenterology nurses.
7.Effects of wearing compression garments on delayed onset muscle soreness based on nuclear magnetic resonance diffusion tensor imaging and physiological&biochemical indices
Miao QIN ; Zidong YANG ; Caizhen CHEN
Chinese Journal of Sports Medicine 2025;44(4):272-280
Objective To explore the intervention effect of tight-fitting equipment on delayed onset muscle soreness(DOMS)after exercise using nuclear magnetic resonance diffusion tensor imaging com-bined with physiological and biochemical indices.Methods Seven healthy adult males participated in a randomized crossover study,and each subject underwent two experiments,with an interval of 7 days,as an elution period to eliminate the effect of the previous experiment.In both experiments,all sub-jects were induced DOMS by finishing 80 times of deep jumps,in 4 groups.In the first experiment,all subjects(the Con group)wore loose clothes during and in 24 hours after the jumping,while in the second one they(the compression sleeve group,Cs)were in full-leg tight leggings.Before,as well as 0 h,24 h and 48 h after the exercise,the muscle soreness,the peak isokinetic torque of the knee extensor muscle group,and the activities of creatine kinase(CK)and lactate dehydrogenase(LDH)were measured.Moreover,before and 24 h after exercise,MRI was performed to measure bi-lateral thigh fractional anisotropy(FA),mean diffusivity(MD),radial diffusivity(RD)and their ei-genvalues(λ1,λ2,λ3).Results The time and group interaction effect for muscle soreness was signifi-cant after exercise(P<0.01),with that of the Cs group significantly lower than the Con group at all three time points after exercise(P<0.01).However,no significant time or group interaction effects were found in the peak isokinetic torque(P>0.05).Moreover,the time and group interaction was sig-nificant in the serum enzyme activity(P<0.05),with CK activity of the Cs group significantly lower than the Con group at 24 h and 48 h after exercise(P<0.05)and LDH activity significantly lower than the latter at 0 h and 24 h after exercise(P<0.05).Meanwhile,MRI showed that the λ2(P<0.05),λ3(P<0.05),and RD(P<0.01)values of quadriceps femoris in the Cs group were significant-ly lower than the Con group at 24 h post-exercise,while the FA values were significantly higher than the latter at the same time point(P<0.05).Conclusion Wearing tight-fitting gear during exercise and within 24 hours after exercise can suppress the elevation of serum CK and LDH activities,and relieve DOMS.Moreover,magnetic resonance diffusion tensor imaging results suggest that it may be realized by limiting the development of localized edema through sustained pressure on the body.
8.Laparoscopic subtotal colectomy with antiperistaltic cecorectal anastomosis versus laparoscopic total colectomy with ileorectal anastomosis for slow transit constipation: a multicenter retrospective cohort study
Yang LUO ; Taotao HOU ; Yifei MU ; Chundi MIAO ; Tingyue GONG ; Jun QIN ; Dongyang WANG ; Dawei SONG ; Hao LI ; Shaolan QIN ; Rong CUI ; Tingfeng WANG ; Ming ZHONG ; Minhao YU
Chinese Journal of Gastrointestinal Surgery 2025;28(12):1426-1433
Objective:To compare postoperative anal function recovery between laparoscopic subtotal colectomy with antiperistaltic cecorectal anastomosis and laparoscopic total colectomy with ileorectal anastomosis for slow transit constipation.Methods:This multicenter retrospective cohort study enrolled patients meeting the following criteria: (1) severe constipation symptoms (<2 bowel movements/week), absent or insignificant defecation urge, abdominal distension, requiring laxatives to maintain bowel movements or laxatives being ineffective; (2) constipation symptoms for over 5 years, ineffective after >2 years of medical treatment, with strong desire for surgery; (3) significantly prolonged colon transit time (>72 hours) without significant gastric or small intestinal transit dysfunction; (4) no organic colonic lesions confirmed by colonoscopy and abdominal CT. Exclusion criteria: (1) patients undergoing open surgery; (2) exclusion of outlet obstruction constipation (e.g., rectocele, rectal prolapse, puborectalis spasm) by functional defecation MRI; (3) comorbid psychiatric disorders; (4) missing clinical data or loss to follow-up (postoperative follow-up <24 months). Based on these criteria, clinical and follow-up data were collected from 220 patients who underwent either laparoscopic subtotal colectomy with antiperistaltic cecorectal anastomosis (LSC group, n = 115) or laparoscopic total colectomy with ileorectal anastomosis (LTC group, n = 105) for slow transit constipation between January 2013 and December 2022. Subjective anal function (Constipation Severity Score and Wexner Fecal Incontinence Score) and objective anal function (positive rate of rectoanal inhibitory reflex [RAIR] and anorectal manometry) were observed preoperatively and at 6, 12, and 24 months postoperatively. Results:No significant differences were found in baseline characteristics between the two groups (all P >0.05). All surgeries were completed successfully without major significant complications. Subjective anal function assessment: At 24 months postoperatively, Constipation Severity Scores decreased significantly compared to preoperative scores in both groups [LSC group: (25.2±2.8) vs. (2.9±1.8), P <0.001; LTC group: (25.8±2.9) vs. (2.8±1.9), P<0.001]. No significant differences were found between the groups at 6, 12, and 24 months postoperatively (all P>0.05). Wexner Fecal Incontinence Scores at 24 months were significantly lower than those at 6 months in both groups [LSC group: (12.9±1.8) vs. (3.9±2.5), P<0.001; LTC group: (12.6±1.8) vs. (5.4±2.4), P<0.001]. Although no significant difference was found at 6 months ( P = 0.190), the LSC group had significantly lower Wexner scores than the LTC group at 12 and 24 months postoperatively (both P < 0.001). Objective anal function assessment: (1) Positive RAIR rate: Preoperative positive RAIR rates were 33.0% (38/115) in the LSC group and 25.7% (27/105) in the LTC group ( P > 0.05). At 24 months, positive rates increased significantly in both groups [LSC: 66.1% (76/115); LTC: 63.8% (67/105)] compared to preoperative rates (both P<0.001), but no significant differences were found between groups at 6, 12, and 24 months (all P>0.05). (2) Resting pressure (RP) and squeeze pressure (SP): No significant differences were found in preoperative RP and SP between groups (all P>0.05). The LSC group had significantly higher RP and SP than the LTC group at 6 and 12 months postoperatively (all P<0.05), but no significant differences were found at 24 months ( P>0.05). Conclusion:Both laparoscopic subtotal colectomy with antiperistaltic cecorectal anastomosis and laparoscopic total colectomy with ileorectal anastomosis are safe for patients with slow transit constipation. However, laparoscopic subtotal colectomy with antiperistaltic cecorectal anastomosis offers superior postoperative anal function recovery.
9.Self-monitoring blood pressure behavior and its influencing factors among residents in Jiangsu Province
MIAO Caiyun ; QIN Yu ; WAN Yanan ; CHEN Lulu ; CUI Lan ; WANG Xiaoli
Journal of Preventive Medicine 2025;37(3):223-227
Objective:
To investigate the self-monitoring blood pressure behavior and its influencing factors among residents in Jiangsu Province, so as to provide the basis for strengthening proactive blood pressure monitoring among residents.
Methods:
Permanent residents aged 35-75 years in six counties (cities, districts), Jiangsu Province, were selected using the stratified cluster sampling method in 2023. Data on basic information, disease history, and self-monitoring blood pressure behavior were collected, height and weight were measured to calculate the body mass index (BMI); and blood glucose and lipid levels were measured. Self-monitoring blood pressure behavior was defined as having measured blood pressure at least once in the past three months. Factors affecting self-monitoring blood pressure behavior were identified using a multivariable logistic regression model.
Results:
A total of 12 475 residents were surveyed, including 5 748 males and 6 727 females, with a male-to-female ratio of 1∶1.17. There were 3 855 residents aged 45-<55 years (30.90%) and 5 511 residents who had self-monitoring blood pressure behaviors (44.18%). Multivariable logistic regression analysis showed that the residents who were males (OR=1.167, 95%CI: 1.081-1.261), lived in rural areas (OR=1.430, 95%CI: 1.321-1.547), aged 45-75 years (45-<55 years, OR=1.384, 95%CI: 1.241-1.543; 55-<65 years, OR=1.397, 95%CI: 1.243-1.570; 65-75 years, OR=1.196, 95%CI: 1.049-1.363), had an annual household income ≥30 000 yuan (30 000-<60 000 yuan, OR=1.190, 95%CI: 1.072-1.321; 60 000-<110 000 yuan, OR=1.330, 95%CI: 1.191-1.485; ≥110 000 yuan, OR=1.746, 95%CI: 1.536-1.984), were overweight (OR=1.170, 95%CI: 1.070-1.280) or obese (OR=1.248, 95%CI: 1.120-1.391), were unaware (OR=1.221, 95%CI: 1.103-1.353) or aware (OR=3.937, 95%CI: 3.575-4.335) of having hypertension, were aware of having diabetes (OR=1.538, 95%CI: 1.354-1.749), and aware of having dyslipidemia (OR=1.265, 95%CI: 1.106-1.447) were more likely to have self-monitoring blood pressure behaviors.
Conclusions
Among the residents aged 35-75 years in Jiangsu Province, 44.18% had self-monitoring blood pressure behavior. Gender, place of residence, age, annual household income, BMI, hypertension, diabetes, and dyslipidemia were identified as influencing factors for self-monitoring blood pressure behavior.
10.Research on The Construction and Application of Multiple Fluorescence Amplification System for Three Kinds of Stains
Yi-Fan BAI ; He-Miao ZHAO ; Jing CHEN ; Hong-Di LIU ; Rui-Qin YANG ; Chong WANG
Progress in Biochemistry and Biophysics 2025;52(4):982-994
ObjectiveA multiplex amplification system was constructed based on the capillary electrophoresis platform for simultaneous detection of saliva, semen, and vaginal secretions using tissue-specific RNA markers. The aim of this study is to identify the tissue origin of suspicious body fluid stains found at crime scenes and determine whether the body fluid stains at the crime scene are one or several types among saliva, semen, and vaginal secretions. MethodsThirty saliva samples, forty semen samples, and forty vaginal secretion samples (half from 2015 and half from 2024) were collected from healthy adult volunteers. Through primer designing, system formulation, and PCR condition optimization, a multiplex fluorescent amplification system was constructed. The specificity, sensitivity, and detection ability for mixed samples of this system were investigated, and it was tested using real crime scene materials. In the primer design stage, to reduce the requirements for RNA template quality, the amplification products were set within 80-300 bp. In the system formulation stage, dominant and subordinate primers were mainly considered. By reducing the concentration of dominant primers and increasing that of subordinate primers, a capillary electrophoresis spectrum with an appropriate peak height ratio was finally obtained. Additionally, gradient experiments were designed to adjust the concentrations of PCR reagents and PCR amplification conditions, and multiple versions of DNA amplification enzymes were optimized to achieve the best experimental results. ResultsThrough statistical analysis, there was no significant difference in the capillary electrophoresis of the 3 types of body fluid samples from the two years (2015 and 2024), demonstrating that the sample preservation method in this study can preserve samples for a relatively long time. The composite amplification system constructed in this study exhibited high specificity for all 3 types of body fluid, with no cross-reactions between the markers of each type of body fluid. The minimum detection thresholds for the 3 types of body fluid reached 0.002 9, 0.001 5, and 0.42 mg/L, respectively. This system also had a high degree of discrimination for mixed samples, especially for semen-saliva mixtures, where each body fluid marker could still be successfully detected when the concentration ratio of semen to saliva was 100:1. Meanwhile, in the two actual cases presented in this article, the application of this composite amplification system performed outstandingly. ConclusionThe composite amplification detection system constructed in this study can achieve the correct screening of saliva, semen, and vaginal secretions, overcoming the problems such as low specificity and sensitivity of marker tests and unbalanced RFU values of each marker in previous studies. The specificity and sensitivity meet the practical work requirements, and the operation is simple. It provides an analytical and identification method for body fluid stains in actual case and is applicable to the identification of the tissue origin of biological evidence at crime scenes involving sexual assault, indecent assault, and other criminal acts. In the future, more types of body fluid markers will be screened to expand the types of body fluids detected by the system, and body fluid-specific cSNP and cInDel genetic markers will be introduced to infer the sources (individuals and types) of mixed and complex stains more accurately.


Result Analysis
Print
Save
E-mail