Objective To investigate the effect of mouse double minute 2 (MDM2) mRNA ASON and mismatched oligonucleotide (ASONM) radiolabeled with 99Tcm on target gene expression in LNCaP cells.Methods The ASON and ASONM targeted to MDM2 mRNA were synthesized and radiolabeled by 99Tcm with the bifunctional chelator of HYNIC.The labeling efficiency,radiochemical purity,stability and molecular hybridization activity were investigated.The different concentrations of 99Tcm-HYNIC-ASON (0,100,500 nmol/L) and 99Tcm-HYNIC-ASONM (500 nmol/L) coated with lipofectamin 2000 were incubated with prostate cancer cells for 24 h,then RT-PCR and Western blot were carried out to assay the MDM2,p53 mRNA and the corresponding protein level.The variables of RT-PCR and Western blot were analyzed using one-way analysis of variance and q test.Results The labeling efficiency of ASON and ASONM were (65.15± 2.05)％ (n=5) and (64.93±2.18)％ (n=5),respectively.The radiochemical purity were both more than 90％.99Tcm-HYNIC-ASON had a good stability and could hybridize to the sense oligonucleotide (SON).The contents of MDM2 mRNA in 0,100,500 nmol/L 99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM groups were 0.458±0.035,0.250±0.026,0.174±0.032,0.463±0.033,respectively,and there were significant differences between each 2 groups except between 0 nmol/L 99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM groups (F=33.69,q =24.32-91.45,all P＜0.01).The average density of MDM2 protein in the 4 groups were 90.712±3.042,71.218±2.915,32.775±3.062,88.121±2.710,respectively (F=235.93,q=6.43-19.14,all P＜0.01; except 0 nmol/L99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM).The contents of p53 mRNA in the 4 groups were 0.185±0.046,0.203±0.040,0.213±0.027,0.163±0.049,respectively(F =2.18,P＞ 0.05).The average density of p53 protein was 33.865 ± 2.213,70.445±2.180,99.025±3.012,38.351±3.271,respectively (F=53.98,q =3.32-6.74,all P＜0.01 ; except 0 nmol/L 99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM).Conclusions The MDM2 antisense probe can accumulate in the prostate cancer cells,and specially hybridize to the MDM2 mRNA and inhibit target gene expression.This novel molecular probe has a promising potential for the diagnosis of prostate cancer at gene level.

Objeetive To explore the value of antisense imaging of 99Tcm-labeled ASON targeting mouse double minute 2(MDM2) mRNA for the diagnosis of human prostate cancer.Methods The ASON targeting MDM2 mRNA and the mismatched oligonucleotide (ASONM) were synthesized and radiolabeled with 99Tcm using the bifunctional chelator HYNIC.The labeling efficiency and radiochemical purity were investigated.Animal models of nude mice bearing human prostate cancer LNCaP were established and divided into 3 groups with 10 mice in each group.99Tcm-HYNIC-ASON,99Tcm-HYNIC-ASONM (study groups) and 99TcmO4-(control group) were injected at the dose of 7.4 MBq through the tail vein,respectively.Tumor imaging was acquired with SPECT and the tumor-to-muscle (T/M) ratio was measured.The data was compared by one-way analysis of variance.Results The labeling efficiencies of ASON and ASONM were (65.15± 2.05) ％ and (64.93±2.18) ％,respectively.Their radiochemical purity was greater than 90％.At 1,4 and 10 h post injection,the T/M ratios of 99Tcm-HYNIC-ASON group were 3.217±0.125,3.749± 0.201 and 4.028±0.186,and those of 99Tcm-HYNIC-ASONM group were 1.579t0.128,1.715±1.140 and 1.683±0.139,and control group 2.146±0.132,1.847±0.124,1.528±0.152,respectively.The T/M ratios in control group and 99Tcm-HYNIC-ASONM group were significantly lower than those in 99Tcm-HYNICASON group at 1,4 and 10 h,respectively (F=213.37-235.41,t=3.527-4.738; all P＜0.01).The T/M ratios of 99Tcm-HYNIC-ASONM group and control group were not significantly different at 1,4 and 10 h (t=2.154,2.287 and 2.236,all P＞0.05).Conclusion The antisense probe of MDM2 can accumulate specifically in prostate cancer tissue in animal models,which might be useful as a non-invasive genetic tool for the early diagnosis of prostate cancer.

Objective To develop multiplex polymerase chain reaction (PCR) combined with reversedotblothy bridization (RDB) method for detection of DNA virus in respiratory samples,and provide a surveillance and rapid diagnosis tool of acute viral respiratory infection.Methods We designed multiple PCR primers and the probes referenced to virus nucleic acid sequences in the National Center for Biotechnology Information (NCBI) database,and fixed specific oligonucleotide probes on the nylon membrane.After multiple PCR amplification of virus DNA of human bocavirus (hBOV),karolinska Institutet (KI),adenovirus (AdV),Washington University polyomaviros (WUPyV),and human parvovirus B19 (HPVBI9),the denaturalized amplification products were hybridized with various specific probes,followed by visualization and analysis of the results.The sensitivity and specificity were tested.At the same time,108 cases of clinical specimens of multiple PCR products were analyzed by reverse spot hybridization detection,and compared to the results of culture method.Results The specific probes of multiple PCR-RDB only hybridized with corresponding amplification products without cross-hybridization reaction with other pathogen.The sensitivity of RDB hybridization was 1 colony-forming units (CFU).The positive rate of 34.26％ (37 cases out of 108 cases) with PCR-RDB method was significandy higher than that 27.78％ (30 cases out of 108 cases) with common test method.Conclusions The multiplex PCR combined with RDB might become a rapid and simple method to detect the DNA virus in respiratory samples,which might be a promising tool for clinical application.

Objective To explore the effects of down regulation of osteopontin(OPN)on the bio- logical behavior of MKN28 and SGC7901 cell lines.Methods OPN siRNA was designed according to the relevant literature and was transfected into the two cell lines.Fluorescent labeling was used to test the transfected efficiency.The down-regulation of OPN protein was measured by Western blot.Real- time PCR was used to test the ratio and time difference of down-regulation of OPN mRNA after siRNA transfection.The biological changes before and after OPN siRNA transfected into these two cell lines were tested by flow cytometry(to test cell cycle and apoptosis)and MTT method(to test the prolifera- tion for the consecutive seven days)and the difference between OPN siRNA transfected or non-transfect- ed cells was compared using mixed model.The capability of moving and invasion of cancer cells were tested by Transwell method and analyzed by t-test.Results The transfected efficiency of OPN siRNA were more than 90% in the two cell lines.OPN mRNA down-regulated to 47% at the 72th hour in SGC7901,while 40% at the 48th hour in MKN28.The expression of OPN protein was both down- regulated after siRNA transfection in the two cell lines.The proliferation decreased after transfected with OPN siRNA both in MKN28 andSGC7901(P

OBJECTIVETo study the methodological techniques in measuring the severity of temporomandibulr disorders (TMD) and in evaluating the effectiveness of therapies in clinic.

METHODSBoth Fricton's Craniomandibular Index (CMI) and Helkimo's Clinical Dysfunction Index were calculated from 60 TMD patients. Inter-rater reliability was tested to assess the consistency in use between different examiners. Fricton's CMI was used to assess the clinical improvement after accepting a treatment in 21 TMD patients diagnosed as acute disk displacement without reduction.

RESULTSCorrelation Coefficient for inter-rater reliability in two groups was 0.879 and 0.939 respectively for Fricton's CMI and 0.744 and 0.838 for Helkimo Clinical Dysfunction Index. Fricton's TMJ dysfunction index was decreased from 0.337 to 0.021 (P < 0.001) and Fricton's CMI was decreased from 0.185 to 0.011 (P < 0.001) after the treatment in 21 TMD patients with disk displacement without reduction.

CONCLUSIONSTo avoid using subjective and descriptive report in assessment of the severity of TMD and the effectiveness of therapies, Fricton's CMI is recommended as an objective criteria which is simple in clinical use, and ease in scoring.

Adolescent ; Adult ; Child ; Female ; Humans ; Male ; Middle Aged ; Severity of Illness Index ; Temporomandibular Joint ; pathology ; Temporomandibular Joint Disorders ; pathology ; Temporomandibular Joint Dysfunction Syndrome ; pathology

OBJECTIVETo compare villus cell culture and karyotype analysis with single nucleotide polymorphism (SNP) microarray technology for the detection of chorionic villus chromosome in patients with retention of abortion.

METHODSForty cases were analyzed with the two methods.

RESULTSChorionic villus culturing was successful in 29 cases, among which 10 were found to have an abnormal karyotypes. For the SNP microarray analysis, all 40 cases were successful, among which 16 were shown to have an abnormal molecular karyotype.

CONCLUSIONSNP microarray technology is highly accurate and specific, which is particularly suitable for the detection of chromosomal deletions or duplications, uniparental disomy, low-percentage mosaicism and other chromosomal abnormalities. It has provided an effective supplement to the conventional chorionic villus culture and karyotype analysis.

Abortion, Missed ; genetics ; Adult ; Chorionic Villi ; chemistry ; Chromosome Aberrations ; Female ; Humans ; Karyotyping ; Male ; Oligonucleotide Array Sequence Analysis ; methods ; Polymorphism, Single Nucleotide ; Pregnancy ; Pregnancy Trimester, First ; genetics

Objective:To explore the level of tibial growth plate chondrocyte mitophagy in young rats with chronic renal failure (CRF) and its effect on chondrocyte apoptosis.Methods:Male 4-week-old Sprague-Dawley rats were randomly divided into two groups according to random number table method: normal control group ( n=20, intragastric administration with distilled water) and CRF group ( n=20, given adenine suspension 150 mg·kg -1·d -1). All the young rats were sacrificed after continuous gavage for 6 weeks. The length of tibia was measured on X ray film, the width of tibia growth plate was measured and compared on histological section, and the apoptosis rate of chondrocytes in growth plate was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. The growth plate chondrocytes of two groups were isolated and cultured to the third generation in vitro, and the apoptosis rate of chondrocytes was detected by TUNEL assay. The co-localization of mitochondria and autophagy lysosomes in chondrocytes was observed by double fluorescence staining. Western blotting was used to detect the level of mitochondrial marker protein translocate of the outer mitochondrial membrane-20 (Tom-20) and autophagy marker light chain-3 protein (LC-3). The mitophagy of growth plate chondrocytes was observed by transmission electron microscope. Results:Compared with the normal control group, the tibia length of CRF group was shorter [(27.32±5.81) mm vs (35.43±3.61) mm, t=5.226, P<0.001], and the relative width of growth plate in histological section was narrower (0.56±0.19 vs 1.00±0.21, t=6.744, P<0.001). The apoptosis rate of chondrocytes in growth plate in CRF group was higher than that in the normal control group (17.2%±4.8% vs 5.1%±3.4%, t=6.505, P<0.001). The apoptosis rate of chondrocytes cultured in vitro in CRF group was higher than that in the normal control group (11.8%±6.2% vs 3.1%±1.2%, t=4.357, P<0.001). The result of double influorescence staining showed that there was co-localization between mitochondria and autophagy lysosomes in CRF group. Western blotting results showed that the levels of LC-3 protein ( t=8.944, P<0.001) and Tom-20 protein ( t=6.708, P<0.001) in CRF group were lower than those in the normal control group. Conclusion:The level of tibial growth plate chondrocyte mitophagy in young rats with CRF increases, which will lead to a decrease in the number of mitochondria, an increase in the apoptosis and a decrease in the number of chondrocytes, and eventually lead to dysplasia of tibia.

Objective:To explore the characteristics and advantages of Whole-Mounting immnunofluorescent staining and laser speckle flow imaging technology in the vascular imaging of mouse ear extended flap.Methods:In this study, total of 25 ICR mice were included.Ten ICR mice were cut off the middle and lateral angiosome to establish an extended flap model, and 3 days later, the changes in the blood supply of the ear flap were observed. The ear area , tissue layer thickness and blood vessel distribution in the healthy side were observed at the same time.Obtain the mouse ears 3 days after modeling, and dissect them into three layers, i.e, the anterior skin layer , the cartilage layer and the posterior skin layer. The distribution and morphology of blood vessels, nerves and monocytes/macrophages in the anterior skin layer were stained and detected by the whole-mount immunofluorescence staining.Ten mice were adopted and an incision was made through the ear horizontally above the bifurcation of the middle angiosome of the mouse ear to establish a delayed extended flap model. Then the blood flow changes in the mouse ear were observed by laser speckle flow imaging andt he blood perfusion values were recorded immediately, 1 d, 2 d, 3 d and 4 d after the operation, respectively.Results:The area of the mouse ear was about 1.3 cm 2 , the thickness was about (0.16±0.04) mm, and the blood was supplied by three vascular bundles: the lateral caudal vascular bundle, the middle vascular bundle and the medial cephalic vascular bundle. The thickness of the anterior and posterior skin and cartilage of the mouse ears were (88±5)μm, (41±3)μm and (29±2)μm, respectively. The whole-mount immunofluorescence staining results clearly showed that the diameter of small vessels in the choke area was (50 ± 6) μm on the third day after modeling. It could be seen that the nerve and artery in mouse ear were in concomitant relationship and the nerve segment attached to the surface of the artery without obvious accompany or clinging to the vein. There were a large number of monocyte macrophages distributed in clusters in the dilated and curved arteries, but they were only scattered outside the artery. Laser speckle flow imaging results showed that there were (6 ± 2) transverse vessels in each auricular flap, and the diameter and blood flow increased significantly in the delayed extended earflap model. Immediately after the operation and at 1d, 2d, 3d, and 4d, the average blood perfusion values of transverse vessels were (92±11) PU, (136±26) PU, (147±27) PU and (176±27) PU, respectively. Conclusions:The Whole-Mounting immnunofluorescent staining and the laser speckle blood flow imaging technology can be used to well observe the blood vessels, nerves, mononuclear macrophages and blood flow perfusion of the mouse extended flap, which can play an important role in the study of blood supply of mouse extended flap.

Objective:To investigate the clinicopathological characteristics of acidophil stem cell pituitary neuroendocrine tumors (PitNET)/adenoma.Methods:Five cases of acidophil stem cell PitNET/adenoma were diagnosed between May 2022 and July 2023 at the Second Hospital of Hebei Medical University, Shijiazhuang, China. The clinicopathological features of the tumor were analyzed by using histology, immunohistochemistry, and electron microscopy. The relevant literature was reviewed.Results:There were 1 male and 4 females, aged from 23 to 69 years. Patient 3 was 55 years old at the time of diagnosis and first surgery, and relapsed 5 years later. The patients′ median age was 32 years. Patients 1 and 5 showed elevated blood prolactin, with various degrees of hormonal symptoms except Patient 3, who showed only tumor compression symptoms. Imaging studies showed that all cases involved the sellar floor. The tumors of Patients 1, 2 and 5 were closely related to the cavernous sinus segment of the internal carotid artery. The tumors exhibited a diffuse growth pattern with chromophobic to slightly acidophilic cytoplasm. A few of tumor cells showed chromophobic cytoplasm. The nucleoli were conspicuous. Intranuclear inclusion bodies and variably-sized clear vacuoles were observed occasionally. Under electron microscope, marked mitochondrial abnormalities were observed, including increased mitochondria number, expanded hypertrophy, and absence of mitochondrial ridge fracture. Some mitochondrial matrices were dense, while some were vacuolated.Conclusions:Acidophil stem cell PitNET/adenoma is a rare type of pituitary adenomas/PitNETs. It often has a more clinically aggressive manner with immature cells, diffuse expression of PIT1, prolactin, and varying degrees of growth hormone expression. Because of the obvious diversity of their clinical hormone status and hormone immune expression, the diagnosis of this type tumor is still a challenge.

Objective To describe the temperature variation within 24 h of the children with congenital heart disease after open heart surgery , the incidence of fever and the relation between fever and clinical outcome, aiming to provide basis for clinical nursing .Methods The convenience sampling method was used to select 200 cases of children with congenital heart disease after open heart operation , collecting body temperature within postoperative 24 hours and clinical outcomes .Results The body temperature of children reached the peak after three hours of open heart operation , then it began to slow down .The fever rate of early postoperative period (within 24 h) was 63.5%.The demographic data of fever group (≥38.0 ℃) and no fever group (<38.0℃) had no significant difference(P>0.05), except for the aspect of clinical diagnosis (χ2 =10.641, P=0.001).The median of ICU stay length, the ventilation time and the total length of hospital stay in fever group and no fever group were 68.50 h and 46.00 h, 20.00 h and 16.00 h, 16.00 d and 12.00 d, which were significantly different (Z =-1.971,-1.998,-3.700, respectively;P<0.05).Conclusions The body temperatures of children with congenital heart disease after open heart operation reach the peak after three hours . More than half of postoperative children will appear postoperative fever , which could affect the clinical outcome .