OBJECTIVE To establish the fingerprint of Pinghuo tea standard decoction and a method for determination of multi-component to clarify the transfer relationship of quantities and quality from pieces and standard decoction. METHODS Fifteen batches of Pinghuo tea standard decoction were prepared and the extract rate was determined； the fingerprint of the preparation was established by using high-performance liquid chromatography（HPLC）； the similarity evaluation and the determination of common peaks were performed， and chemometric analysis was performed； the same method was used to determine the content of indicator components and the transfer rate was calculated. The chromatographic column was Venusil C18 column with mobile phase consisted of acetonitrile-0.1% phosphoric acid solution （gradient elution）； the column temperature was 30 ℃， and the detection wavelengths were 238 nm （0-37 min， 85-102 min） and 330 nm （37-85 min） at a flow rate of 1.0 mL/min with an injection volume of 10 μL. RESULTS The similarity of HPLC fingerprints for 15 batches of Pinghuo tea standard decoction was not lower than 0.968. A total of 24 common peaks were calibrated and 9 peaks were recognized， which were as follows neochlorogenic acid （peak 3）， chlorogenic acid （peak 6）， geniposide （peak 9）， glycyrrhizin （peak 10）， galuteolin （peak 11）， isochlorogenic acid A （peak 14）， luteolin （peak 21）， kaempferol （peak 23） and glycyrrhizic acid （peak 24）. Cluster analysis， principal component analysis and orthogonal partial least squares discriminant analysis showed consistent results， all of which could classify the 15 batches of samples into three categories. The linear range of indicator components in 15 batches of Pinghuo tea standard decoction， such as geniposide， luteolin， isochlorogenic acid A， glycyrrhizin， and glycyrrhizic acid， were 0.020 580-0.411 600， 0.001 617-0.080 850， 0.006 076-0.607 600， 0.005 125-0.071 740， and 0.017 288-0.432 200 mg/mL， respectively； RSDs of precision， repeatability， stability and recovery rate tests were all not higher than 4% （n＝6）. The mass fractions ranged 3.227 9-10.002 2， 0.297 4-0.554 6， 3.350 1-6.159 6， 0.720 6-1.073 3， 2.003 1-3.030 1 mg/g； transfer rates from the pieces and standard decoction were 19.762 8%-35.840 5%， 12.123 3%-21.254 0%， 46.097 2%-82.869 4%， 58.708 8%-91.629 6%， 39.114 3%-63.710 6%. The transfer rates of the extract from 15 batches of Pinghuo tea standard decoction ranged from 61.15%-84.68%. CONCLUSIONS Established HPLC fingerprint and content determination methods in this study are simple and accurate， which can provide reference for the quantitative value transfer study， quality control， clinical application and the development of subsequent formulations of Pinghuo tea standard decoction.

OBJECTIVES: To reprogram human placental fibroblasts (HPFs) into chemically induced epithelioid-like cells (ciEP-Ls) using a combination of small-molecule compounds. METHODS: HPFs cultured under normoxic conditions were identified using immunofluorescence assay, PCR and chromosomal karyotyping. Under hypoxic conditions (37 ℃, 5% O₂), HPFs were cultured in a medium containing small-molecule compounds C6TSEDRVAJZ (CHIR99021, 616452, TTNPB, SAG, EPZ5676, DZNep, Ruxolitinib, VTP50469, Afuresertib, JNK-IN-8, and EZM0414), and the cell morphology was observed daily. The expression levels of epithelial cell markers in the induced cells were detected by immunofluorescence, Western blotting and PCR. Chromosomal karyotyping of the induced cells was performed and the induction efficiency was calculated. RESULTS: Before induction, HPFs showed positive expressions of fibroblast surface markers CD34 and vimentin and were negative for epithelial surface markers. PCR results showed high expressions of fibroblast-specific genes S100A4 and COL1A1 in HPFs with a normal human diploid karyotype. After one day of induction, the HPFs underwent morphological changes from a multinodular spindle shape to a round or polygonal shape, which was morphologically characteristic of ciEP-Ls. On day 4 of induction, the cells exhibited high expressions of the epithelial cell markers E-cadherin and Lin28A. RT-qPCR results also showed that the cells expressed the epithelial markers Smad3, GLi3, PAX8, WT1, KRT19, and KRT18 with significantly down-regulated expressions of all the fibroblast surface markers and a normal human diploid karyotype. The reprogramming efficiency of HPFs into ciEP-Ls ranged from (64.53±2.8)% to (68.10±3.6)%. CONCLUSIONS The small-molecule compound combination C6TSEDRVAJZ is capable of inducing HPFs into ciEP-Ls under hypoxic conditions with a high induction efficiency.
Humans ; Fibroblasts/drug effects* ; Pregnancy ; Female ; Cell Differentiation/drug effects* ; Pyrimidines/pharmacology* ; Placenta/cytology* ; Cells, Cultured ; Pyridines/pharmacology* ; Pyrazoles/pharmacology* ; Epithelial Cells/cytology*

Objective To improve the efficiency of induced differentiation of primitive neural epithelial cells derived from human induced pluripotent stem cells(hiPSCs-NECs)into functional midbrain dopaminergic progenitor cells(DAPs).Methods HiPSCs were cultured in mTeSRTM medium containing DMH1(10 μmol/L),SB431542(10 μmol/L),SHH(200 ng/mL),FGF8(100 ng/mL),purmorphamine(2 μmol/L),CHIR99021(3 μmol/L),and N2(1%)for 12 days to induce their differentiation into primitive neuroepithelial cells(NECs).The hiPSCs-NECs were digested with collagenase IV and then cultured in neurobasal medium supplemented with 1%N2,2%B27-A,BDNF(10 ng/mL),GDNF(10 ng/mL),AA,TGF-β,cAMP,and 1%GlutaMax in the presence of different concentrations of Rho kinase inhibitor Y27632,and the culture medium was changed the next day to remove Y27632.Continuous induction was performed until day 28 to obtain DAPs.Results Human iPSCs expressed the pluripotency markers OCT4,SOX2,Nanog,and SSEA1 and were positive for alkaline phosphatase staining.The hiPSCs-NECs were obtained on day 13 in the form of neural rosettes expressing neuroepithelial markers SOX2,nestin,and PAX6.In digested hiPSCs-NECs,the addition of 5 μmol/L Y27632 significantly promoted survival of the adherent cells,increased cell viability and the proportion of S-phase cells(P<0.01),and reduced the rate of apoptotic cells(P<0.05).On day 28 of induction,the obtained cells highly expressed the specific markers of DAPS(TH,FOXA2,NURR1,and Tuj1).Conclusion Treatment with Y27632(5 μmol/L)for 24 h significantly promotes the survival of human iPSCs-NECs during their differentiation into DPAs without affecting the cell differentiation,which indirectly enhances the efficiency of cell differentiation.

Objective To improve the efficiency of induced differentiation of primitive neural epithelial cells derived from human induced pluripotent stem cells(hiPSCs-NECs)into functional midbrain dopaminergic progenitor cells(DAPs).Methods HiPSCs were cultured in mTeSRTM medium containing DMH1(10 μmol/L),SB431542(10 μmol/L),SHH(200 ng/mL),FGF8(100 ng/mL),purmorphamine(2 μmol/L),CHIR99021(3 μmol/L),and N2(1%)for 12 days to induce their differentiation into primitive neuroepithelial cells(NECs).The hiPSCs-NECs were digested with collagenase IV and then cultured in neurobasal medium supplemented with 1%N2,2%B27-A,BDNF(10 ng/mL),GDNF(10 ng/mL),AA,TGF-β,cAMP,and 1%GlutaMax in the presence of different concentrations of Rho kinase inhibitor Y27632,and the culture medium was changed the next day to remove Y27632.Continuous induction was performed until day 28 to obtain DAPs.Results Human iPSCs expressed the pluripotency markers OCT4,SOX2,Nanog,and SSEA1 and were positive for alkaline phosphatase staining.The hiPSCs-NECs were obtained on day 13 in the form of neural rosettes expressing neuroepithelial markers SOX2,nestin,and PAX6.In digested hiPSCs-NECs,the addition of 5 μmol/L Y27632 significantly promoted survival of the adherent cells,increased cell viability and the proportion of S-phase cells(P<0.01),and reduced the rate of apoptotic cells(P<0.05).On day 28 of induction,the obtained cells highly expressed the specific markers of DAPS(TH,FOXA2,NURR1,and Tuj1).Conclusion Treatment with Y27632(5 μmol/L)for 24 h significantly promotes the survival of human iPSCs-NECs during their differentiation into DPAs without affecting the cell differentiation,which indirectly enhances the efficiency of cell differentiation.

Objective To observe the effect of extracorporeal shockwave therapy (ESWT) in regulating endothelial cell phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression on lower ex-tremity vascular lesion and its possible mechanism.Methods Twenty-four 2-month-old healthy male SD rats were randomly divided into the three groups:control group (group A),diabetes angiopathy group (group B),diabetes angiopathy+ESWT group (group C).The group B and C were fed with high fat and high sugar and intraperitoneally injected with streptozotocin 60 mg/kg to establish the rat model of diabetes vascular lesion. The group C received ESWT at 1 week (T1),2 weeks (T2),3 weeks (T3) and 4 weeks (T4) after modeling,and the blood stream velocity of rat femoral artery vascular lesion area and vascular internal diameter were measured at T4 by ultrasound.At the end of ESWT,the rats were immediately killed for taking their femoral arteries and gastrocnemius.The structures of the femoral arteries in each group were observed under electron microscopy.Western blot was used to detect the expression levels of PTEN,PI3K and Akt proteins,while qRT-PCR was used to detect the expression levels of PTEN mRNA.Immunofluorescence was used to detect the expression level of CD31 in gastrocnemius muscle .Results The peak systolic flow velocity and end-dias-tolic flow velocity of femoral artery at T4 in group B and C were significantly lower than those in group A (P<0.05),but group C was higher than group B (P<0.05).The internal diameter of femoral artery had no statistical difference among 3 groups (P>0.05).The PTEN expression level in group B and group C was sig-nificantly lower than that in group A (P<0.05),while group C was higher than group B (P<0.05).The ex-pression levels of PI3K and Akt in group B were higher than those in group A (P<0.05),and group C was lower than group B (P<0.05).The PTEN mRNA expression level in group B and group C was significantly lower than that in group A (P<0.05),but group C was higher than group B (P<0.05).Under electron mi-croscopy,it was observed that after ESWT,the endothelial cell damage in group C was obvious when com-pared with group B.The CD31 expression level in group B and group C was significantly lower than that in group A (P<0.05),but group C was higher than group B (P<0.05).Conclusion ESWT could improve the vascular function,increase the peak velocity during systolic period of femoral artery in diabetes rats and im-prove the microvessel density of gastrocnemius muscle by up-regulating PTEN in lower extremity artery and down-regulating PI3K and Akt in diabetes rats.

BACKGROUND:Resistance to the inflammatory response is an important part of promoting the repair of damaged tissue and improving the local inflammatory response caused by medical bio-implant materials has been a key issue to be addressed in recent years. OBJECTIVE:To summarize the anti-inflammatory effects of common metal ions and related molecular mechanisms to provide some theoretical references for improving the early inflammatory response of hosts caused by bio-implant materials. METHODS:A computer search of the relevant literature in PubMed,Web of Science,CNKI and WanFang databases was conducted using"metal ions,magnesium ion,zinc ion,silver ion,copper ion,inflammation,anti-inflammatory effects,oxidative stress,immunoregulation,signaling pathways"as Chinese and English search terms.Preliminary screening was conducted by reading the titles and abstracts.Finally,80 papers were included for result analysis and summary. RESULTS AND CONCLUSION:(1)Metal ions such as magnesium,zinc,silver and copper have a good anti-inflammatory effect.The strength of this anti-inflammatory effect is strongly correlated with the dose and duration of action.In the future,consideration can be given to controlling the release rate of ions and adjusting the appropriate therapeutic concentration to achieve the best anti-inflammatory effect.(2)Magnesium ions and zinc ions exhibit excellent anti-inflammatory activity,with magnesium ions often being beneficial in anti-inflammatory therapy in the form of compounds such as magnesium sulfate and zinc ions regulating the body's inflammatory response with zinc feed as the main source of zinc supplementation.(3)Silver and copper ions have some anti-inflammatory effects,but are still predominant for their excellent antibacterial activity,mainly in the form of nanoparticles and bio-coatings.(4)Magnesium and zinc metal ions can be combined with natural extracts to form complexes to exert anti-inflammatory effects,and this method has the advantage of being inexpensive and widely available and is a sustainable and green approach,which is worthy of clinical promotion.(5)Metal ions such as magnesium,zinc,silver and copper exert anti-inflammatory effects by reducing host oxidative stress damage,modulating immune cells and inhibiting inflammatory signaling pathways such as nuclear factor-κB,Toll-like receptor,STAT3 and NOD.(6)The molecular mechanism related to the anti-inflammation of metal ions is a complex network,which is not the effect of a single pathway,but should be a combination of multiple signaling pathways.There are still many potential mechanisms that have not yet been explored,and more systematic elucidation of the interconnections between various signaling pathways is needed in the future.

Objective:To establish an automated labeling method of 18F-AlF-1, 4, 7-triazocyclohexane-1, 4, 7-triacetic acid- D-Phe1-Tyr3-Thr8-octreotide (NOTATATE) and perform neuroendocrine tumor (NET) imaging. Methods:Based on the GE-FASTLab2 synthesis module, 18F-AlF-NOTATATE was automatically prepared by one-step chelation labeling with aluminum fluoride, and its labeling conditions were optimized. The product quality was analyzed. One patient (male, 47 years old) with lower rectal segment NET and one patient (female, 52 years old) with pancreatic NET underwent 18F-AlF-NOTATATE PET/CT imaging. Results:18F-AlF-NOTATATE was successfully prepared with a total synthesis time of 35 min. The optimized radiochemical yield was (23.8±3.1)% (without decay correction, n=3), the radioactivity was (4.63±0.68) GBq, and the radiochemical purity was >95%. The stability was good, and the product quality met the requirements. 18F-AlF-NOTATATE showed clear imaging in the patient with rectal segment NET, with SUV max of 13.3 and tumor/liver ratio of 3.3. Metastatic lesions in the liver, lymph nodes, and ribs showed high SUV max and tumor/liver ratios. The imaging of the pancreatic NET patient showed an abnormal increase in local radioactive uptake at the uncinate process of the pancreatic head, with SUV max of 5.6 and SUV max of 6.3 and the tumor/liver ratio of 2.3 after 2-hours imaging. Conclusions:Using the GE-FASTLab2 synthesis module, 18F-AlF-NOTATATE can be prepared with high activity. The preparation is simple, the method is stable, and the product has high radiochemical purity. 18F-AlF-NOTATATE exhibits good imaging performance in NET patients, providing valuable information for diagnosis, treatment, and prognosis evaluation.

Objective:To evaluate the clinical application potential of a novel prostate specific membrane antigen (PSMA) targeted PET tracer 68Ga-PSMA-NYM032 in patients diagnosed initially with prostate cancer. Methods:A total of 63 patients (age (68.7±8.7) years) with suspected prostate cancers who received 68Ga-PSMA-NYM032 PET/CT imaging in Affiliated Hospital of Jiangnan University between March 2022 and January 2023 were enrolled prospectively. The diagnostic efficiency of 68Ga-PSMA-NYM032 PET/CT imaging was evaluated in a patient-centered manner. The ROI was drawn to obtain SUV max by semi-quantitative analysis with visual analysis, and the diagnostic threshold of SUV max was obtained by ROC curve analysis. The correlations of SUV max in primary foci with total prostate specific antigen (tPSA) and Gleason score (GS) were analyzed by Spearman rank correlation analysis. Based on the D′Amico risk stratification (prostate specific antigen (PSA)>20 μg/L and ≤20 μg/L, GS>7 and ≤7), the detection rates of metastases by 68Ga-PSMA-NYM032 PET/CT imaging in different stratifications were analyzed by Fisher exact test, and the differences between SUV max of metastases in different stratifications were determined by Mann-Whitney U test. Results:The accuracy of 68Ga-PSMA-NYM032 PET/CT imaging was 92.06%(58/63), the sensitivity was 96.55%(28/29), the specificity was 88.24%(30/34), the positive predictive value was 87.50%(28/32), the negative predictive value was 96.77%(30/31), and the optimal SUV max threshold was 6.9. 68Ga-PSMA-NYM032 showed varying degrees of high uptake in the primary foci of prostate cancer, and SUV max were positively correlated with tPSA and GS ( rs values: 0.657, 0.592, P values: <0.001, 0.001). Stratified analysis showed a statistically significant difference in the detection rate of bone metastases by 68Ga-PSMA-NYM032 PET/CT between the GS>7 and GS≤7 subgroups (9/17 vs 1/12; P=0.019), while no statistical significances were observed in the detection rates of bone metastases or lymph node metastases of another subgroups (all P>0.05). In addition, none of the differences in SUV max of metastases in patients with different stratifications were statistically significant ( z value: from -1.57 to -0.50, all P>0.05). Conclusions:68Ga-PSMA-NYM032 PET/CT imaging has good diagnostic efficacy for prostate cancer, and it may provide a new strategy for the precise diagnosis and treatment of prostate cancer. Besides, GS stratification may affect the detection rate of bone metastases by 68Ga-PSMA-NYM032 PET/CT imaging.

A triple-transgenic (tyrosine hydroxylase/dopamine decarboxylase/GTP cyclohydrolase 1, TH/DDC/GCH1) bone marrow mesenchymal stem cell line (BMSCs) capable of stably synthesizing dopamine (DA) transmitters were established to provide experimental evidence for the clinical treatment of Parkinson's disease (PD) by using this cell line. The DA-BMSCs cell line that could stably synthesize and secrete DA transmitters was established by using the triple transgenic recombinant lentivirus. The triple transgenes (TH/DDC/GCH1) expression in DA-BMSCs was detected using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. Moreover, the secretion of DA was tested by enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). Chromosome G-banding analysis was used to detect the genetic stability of DA-BMSCs. Subsequently, the DA-BMSCs were stereotactically transplanted into the right medial forebrain bundle (MFB) of Parkinson's rat models to detect their survival and differentiation in the intracerebral microenvironment of PD rats. Apomorphine (APO)-induced rotation test was used to detect the improvement of motor dysfunction in PD rat models with cell transplantation. The TH, DDC and GCH1 were expressed stably and efficiently in the DA-BMSCs cell line, but not expressed in the normal rat BMSCs. The concentration of DA in the cell culture supernatant of the triple transgenic group (DA-BMSCs) and the LV-TH group was extremely significantly higher than that of the standard BMSCs control group (P < 0.000 1). After passage, DA-BMSCs stably produced DA. Karyotype G-banding analysis showed that the vast majority of DA-BMSCs maintained normal diploid karyotypes (94.5%). Moreover, after 4 weeks of transplantation into the brain of PD rats, DA-BMSCs significantly improved the movement disorder of PD rat models, survived in a large amount in the brain microenvironment, differentiated into TH-positive and GFAP-positive cells, and upregulated the DA level in the injured area of the brain. The triple-transgenic DA-BMSCs cell line that stably produced DA, survived in large numbers, and differentiated in the rat brain was successfully established, laying a foundation for the treatment of PD using engineered culture and transplantation of DA-BMSCs.
Rats ; Animals ; Dopamine ; Parkinson Disease/metabolism* ; Mesenchymal Stem Cells/metabolism* ; Cell Line ; Brain/metabolism* ; Cell Differentiation ; Mesenchymal Stem Cell Transplantation

Objective:To evaluate the value of 18F-FDG PET/CT in the diagnosis and treatment of primary breast lymphoma (PBL). Methods:Clinical data and 18F-FDG PET/CT imaging data of 6 patients (all females, age 46-79 years) with pathologically diagnosed primary breast diffuse large B cell lymphoma (PB-DLBCL) in Xishan People′s Hospital of Wuxi City and Affiliated Hospital of Jiangnan University from July 2015 to October 2021 were analyzed retrospectively. A total of 10 18F-FDG PET/CT scans were done for primary staging (6 scans of 6 patients), evaluation of treatment response (3 scans of 2 patients), and recurrence detection (1 scan of 1 patient). 18F-FDG PET/CT image analysis was performed qualitatively (visually) and semi-quantitatively (SUV max). Treatment response was evaluated by Deauville scores. Results:All 6 patients were diagnosed pathologically as PB-DLBCL (3 patients by core needle biopsy, 3 patients by biopsy after lumpectomy). All 6 patients were staged using baseline 18F-FDG PET/CT before chemotherapy. For 3 patients diagnosed by core needle biopsy, baseline 18F-FDG PET/CT showed unilateral breast lesion with high FDG uptake (SUV max: 23.0, 52.9, and 33.6). For 3 postoperative patients, baseline 18F-FDG PET/CT showed flocculent soft tissue density in the operative area with low FDG uptake (SUV max: 3.4, 2.2 and 2.0). Patient No.2 showed a large left breast mass with left axillary lymph node involvement by baseline PET/CT, and multiple nodular uptakes in bilateral breast (Deauville score of 4) after 4 courses of chemotherapy and negative result (Deauville score of 1) after 3 courses of new chemotherapy regimens by PET/CT. Patient No.4 showed right breast lesion and right axillary lymph nodes by routine preoperative imaging examination, but left breast lesion by postoperative PET/CT. According to the results of 18F-FDG PET/CT, patient No.4 was with complete response (Deauville score of 1) after treatment, but recurrence (Deauville score of 5) occurred after 7 months follow-up. Conclusion:18F-FDG PET/CT can play an important role in every step of management (diagnosis and staging, treatment response evaluation and detection of recurrence) in patients with PB-DLBCL.