Preliminary application experience of disk microfluidic chip for detecting CALR gene mutation in patients with cerebral infarction / 中华检验医学杂志

Objective:To establish a disk (CD) microfluidic chip detection platform for the rapid detection of CALR-1 and CALR-2 mutations in patients with cerebral infarction, and summarize its clinical application value.Methods:Based on microfluidic technology and loop mediated isothermal amplification technology, a CD microfluidic chip detection platform for simultaneous detection of CALR-1 and CALR-2 gene mutations were established, and the sensitivity, specificity, repeatability and accuracy of the platform were verified. A total of 124 patients with cerebral infarction treated in Huashan Hospital, Shanghai Medical College, Fudan University from November 2019 to March 2021 were prospectively selected into the experimental group; and 80 healthy subjects were included in the control group. The CALR-1 and CALR-2 gene mutations in anticoagulant peripheral blood samples were detected by the CD microfluidic chip. Each chip could detect 4 samples at the same time and synchronously detect 3 indexes of each sample. The detection results could be obtained after isothermal amplification for 40 min. At the same time, sequencing method was used to verify the test results, and the consistency of the results of the two detection methods was compared.Results:Using this CD microfluidic chip platform, the synchronous amplification of 3 indexes in the sample could be completed within 40 min without the need of thermal circulation, and the whole detection process of the sample could be completed within 60 min. For samples with a high concentration of target nucleic acid, typical positive signals could be visualized after amplification for 10 min, and the test results would be available within 30 minutes after receiving the samples. The detection sensitivity of CD microfluidic chip method for CALR-1 and CALR-2 mutation load concentration was 1.0% and 0.5% respectively. Nonspecific amplification was not observed for the non-target nucleic acid samples, indicating the high specificity of this method. The coincidence rates of intra and inter batch repeatability were 100% (20/20) respectively. Two samples with CALR gene mutation were found in the cerebral infarction group, both of which were CALR-1 mutations (L367fs*46). There was no CALR-1 or CALR-2 mutation in the control group. The detection results of CD microfluidic chip method were completely consistent with the sequencing verification results (100% [204/204]).Conclusions:The CD microfluidic chip method could be used for the detection of CALR-1 and CALR-2 gene mutations in clinical samples of patients with cerebral infarction. This method has the advantages of high detection sensitivity, good detection specificity, fast detection speed and high detection flux, which is helpful to clarify the etiology of patients with cerebral infarction.

Factors accounting for HIV antibody test false positive results in patients with rheumatoid arthritis / 中华检验医学杂志

Objective To investigate if immunological factors associated with the false HIV screening test results in RA.Methods Subjects who attended the Rheumatology Outpatient Clinic -Internal Medicine Unit of Guanghua Integrative Hospital , from October 2013 to October 2014, who met the American College of Rheumatology /European League Against Rheumatism Criteria for RA were recruited for the study . 100 subjects with RA were recruited.Each patient underwent clinical examination and blood sampling for assessment of serum HIV screening test and Rheumatoid factors ( RF-IgA, -IgG, -IgM) and anti-cyclic citrullinated protein antibodies (anti-CCP) were purified from the plasma and detected by ELISA , Samples were collected and processed using standard protocols and were stored in the same freezer before analysis . RA patients were divided into two groups based on the titters of RF and anti -CCP:RF 300 U/ml /anti-CCP >500 U/ml group.HIV screening tests were determined by three methods: ELISA、Immuno-colloidal Golden Method and ECLIA.The positive results were confirmed by the Changning Centers for Disease Control , Shanghai through western -blotting test.Results 100 samples detected by ELISA and Immuno-colloidal Golden Method were given negative results , 16 positive results existed in ECLIA group.There were1,12,3 positive cases in RF-IgM 300 U/ml group(2.7%,32.4%,13.6%;P 500 RU/ml groups there were 2 and 4 positive results(4.7%,24.6%;P <0.01).Conclusions Different HIV screening test methods would give different results , according to operation requirement using second method to determine the HIV screening result.HIV False-positivity was associated with the titers of anti -CCP and RF in RA.

ClinicaI significance of T follicuLar helper cells in rheumatoid arthritis / 国际检验医学杂志

Objective To investigate the percentages of T follicuLar helper cells (Tfh)and interleukin-21(IL-21)in the plasma of patients with rheumatoid arthritis,and the immunological mechanism of Tfh cells in the development of rheumatoid arthritis. Methods According to ACR and DAS28,patients with rheumatoid arthritis were divided into low,moderate and high activity group.The percentages of CD3 + CD4 + CXCR+ 、CD3 + CD4 + ICOS+ and CD3 + CD4 + CXCR+ ICOS+ Tfh cells were detected by Flow Cytometry.While the levels of IL-21 、RF-IgM and anti-CCP in plasma were measured by ELlSA test.The analysis was performed by t-test and Spearman′s correlation analysis.Results The expression of CD3 + CD4 + CXCR+ ICOS+ Tfh cells in PBMCs of rheu-matoid arthritis was significantly higher than that in the normal controls(P <0.05).Meanwhile the results of the three rheumatoid arthritis groups(low,moderate and high activity groups)showed that the expression of Tfh increased accordingly(P <0.05).The expression of Tfh in rheumatoid arthritis was positively related with the levels of IL-21 ,ESR,CRP,RF and anti-CCP respectively. Conclusion The expression of Tfh and IL-21 increases significantly and is closely related to the disease activity in rheumatoid ar-thritis.The results indicate that the abnormality of Tfh may play an important role in the pathogenesis of rheumatoid arthritis.

Antitumor effects of radioiodine labeled KH901 on nude mice bearing hepatoma / 生物医学工程学杂志

In order to evaluate the biological activity in vitro and the antitumor effects of 131I-conditionally replicating oncolytic adenovirus KH901 on HepG2 human hepatoma xenografts, the leves of GM-CSF expression were determined by ELISA method. A panel of tumor and normal cells was infected with recombinant adenovirus KH901 at MOI of 10 PPC. The medium was harvested to determine the bioactivity of GM-CSF after 24 hours. Nude mice bearing HepG2 human hepatoma xenografts were given 131-KH901. Antitumor effects were assessed using endpoints of tumor growth delay. The data showed that after 24 hours 131-KH901 replicated hugely in tumor cells and produced significant amount of GM-CSF 183.27 +/- 6.90 pg/ml, while producing very small amount of GM-CSF 20.44 +/- 0.77 pg/ml in normal cells. In the treatment of tumor, 131I-KH901 showed higher restraint rate (71.3%) compared to 131I (22.7%) or KH901 (52.7%). Therefore, 131-KH901 can inhibit the growth of human hepatoma cell in nude mice and it may be a potential drug for treating liver cancer.

Study on apoptosis of the HCT116 cells mediated by cisplatin and its mechanisms / 中国综合临床

Objective To study the apoptosis mediated by cisplatin on human colorectal tumors (HCT116) cell line and its mechanisms in vitro. Methods The apoptosis levels of HCT116 cells mediated by cisplatin at vari-ous time and in different concentration were measured by M30-ApoptosisTM -ELISA-kits and flow cytometry assay. The expressions of the protein p53,p21 and Bcl-2 were assessed through Western-Blotting. Results Cisplatin in-hibited the proliferation in a time-and dose -dependant manner ( F = 1129. 383, P = 0. 000 and F = 125. 267, P = 0. 000, respectively). The sub-G1 peak detected by flow cytometry at 24,48,72 hours of the apoptosis rates showed a significant difference between the experimental group and the control group (χ2= 5. 669,14.110,12. 221, P = 0. 010,0.003,0. 000,respectively). We found significant differences on the HCT116 cell growth between different time under cisplatin effect (χ2 = 14.008 ,P =0. 003 ). There were significant difference on the CK18-Asp237-Asp396 re-leased by HCT116 cell between different casplatin concentration (F =48. 667 ,P =0.000) as well as different times ( F = 1194. 394, P = 0.000). The Western-Blotting results indicated that the expression level of the p53 ( t = 9.873, -2.906,7. 229,2.776,P =0.000,0. 007,0. 000,0. 011 ) and p21 (t = - 10. 692, - 8. 867, - 15. 063, - 16.281, P = 0. 000,0.001,0.000,0.000 ,respectively) increased gradually, while there are no effect on the expression of the protein Bcl-2(t=1.429,2.011,2.247,2.001,P=0. 178,0.069,0.053,0.062,respectively). Conclusions Cis-platin can induce the apoptosis on HCT116 cells and thus inhibit the reproduction of tumor cells by recovering the function of p.53.

Iodination conditions of KH901, a tumor-specific oncolytic recombinant adenovirus, and its 125I-labeled compounds biodistribution in animals / 生物医学工程学杂志

In this research was developed high efficiency method using 125I for directly labeling KH901, a tumor-specific oncolytic recombinant adenovirus, biodistribution of 125I-labeled compound in normal mice was investigated. 125I-KH901 was prepared by N-bromosuccinimide labeling method to find the optimal ratio of labeling response. The compounds were isolated and purified by Sephadex-G10 agarose and the radiochemical purity of compounds was analyzed by paper chromatography. The radioactivity biodistribution in mice was measured at different times after caudal vein injection with 0.1ml 125I-KH901. The labeling yield of 125I-KH901 was 78% and the radiochemical purity was 95% after purification by Sephadex-G10 agarose. Biodistribution revealed that the uptake of 125I-KH901 in liver was higher than in other organs at all time points of the experiment. 125I-KH901 was mainly concentrated in liver, kidneys, spleen and lung. It can be seen that N-bromosuccinimide labeling method is an optimal method with simple steps and high labeling yield in labeling KH901 with 125I. 125I-KH901 has a biodistribution trait which is an advantage to treating liver tumors.

The diagnostic value of 99mTc-MIBI myocardial perfusion imaging for coronary artery disease: a systematic review / 生物医学工程学杂志

This review aims to evaluate the quality of studies assessing the value of 99mTc-MIBI myocardial perfusion imaging in the diagnosis of coronary artery disease. OVID (1956 to 2006), CBMdisc (1978 to 2006), CNKI (2005 to 2006) and VIP (2005 to 2006) for relevant studies in English and Chinese were searched and identified. Quality assessment of diagnostic accuracy studies (QUADAS) items were used. Studies were classified and Meta-disc software was used to analyze sensitivity, specificity, positive likelihood ratio and negative likelihood ratio for the pooled analysis and heterogeneity test, then Asymmetric SROC curves were drawn for those without heterogeneity. In 29 articles included, the results of the pooled analysis showed that, as for rest, exercise and drug myocardial perfusion imaging, the pooled LR + were 2.209, 4.334 and 5.508, the pooled LR- were 0.224, 0.141 and 0.195, and for dipyridamole myocardial perfusion imaging, the pooled LR+ and LR- were 5.031 and 0.193, respectively. Besides, for stress myocardial perfusion imaging among the patients without myocardial infarction history, the pooled LR+ and LR- were 6.176 and 0.199, respectively. The biases from the 29 studies were mainly due to diagnostic test results review bias; variations were probable and were correlated with the spectrum of disease and inclusion criteria; the quality of report was moderate. The conclusion is that 99mTc-MIBI stress MPI, especially dipyridamole MPI, is valuable for diagnosing coronary artery disease.

A comparison on radiochemical behavior and biological property of antisense oligonucleotide labeled with technetium-99m by two methods: NHS-MAG3 versus SHNHP / 生物医学工程学杂志

This study was undertaken to explore and compare the radiochemical behavior and biological property of antisense oligonucleotide (ASON) labeled with Technetium-99m using two methods: N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycline (NHS-MAG3) versus hydrazino nicotinamide derivative (SHNH). After SHNH and NHS-MAG3 were synthesized, ASON was labeled with Technetium-99m using SHNH and NHS-MAG3 as a bifunctional chelator, separately. The stability in vivo and in vitro, the combination with plasma albumen of rabbit, the biodistribution in BALB/ C mice and the HT29 cellular uptake were compared between labeled compound 99mTc-SHNH-ASON, using SHNH as a bifunctional complex reagent, and 99mTc-MAG3-ASON, using NHS-MAG3 as a bifunctional chelator. The results revealed that the labeling rate and the stability of 99mTc-MAG3-ASON were evidently higher than that of 99mTc-SHNH-ASON (P < 0.05), the combination rate of 99mTc-MAG3-ASON with plasma albumen was markedly lower than that of 99mTc-SHNH-ASON (P < 0.05); the biodistribution of 99mTc-MAG3-ASON was markedly lower than that of 99mTc-SHNH-ASON in blood, heart, stomach and intestines (P < 0.05), slightly lower than that of 99mTc-SHNH-ASON in liver and spleen (P > 0.05), and markedly higher than that of 99mTc-SHNH-ASON in kidney (P < 0.05); the HT29 cellular uptake rates of 99mTc-MAG3-ASON was markedly higher than that of 99mTc-SHNH-ASON (P < 0.05). Therefore, the radiochemical behavior and biological property of 99mTc-MAG3-ASON labeled using NHS-MAG3 is better than that of 99mTc-SHNH-ASON labeled using SHNH.

Progess on research of herpes simplex virus type 1 mutants for cancer therapy / 生物医学工程学杂志

For a long time past viruses have been recognized as being tumoricidal. At present, researchers are still pursuing studies and constructing more suitable oncolytic viruses for treating different malignant tumors. Herpes simplex virus type 1 (HSV-1) has been known as the most potential oncolytic virus among all the viruses. In this overview, we summarize the current situation of oncolytic viruses, the biology of HSV-1, its construction and application of its recombinant, and we debate on the feasibility and prospect of HSV-1 mutants labeled with radionuclides for cancer therapy.

Pharmacokinetics of injection of iodine-131 labelling MEI-TUO-XI monoclonal antibody in human body / 生物医学工程学杂志

To study pharmacokinetics of injection of iodine-131 labelling MEI-TUO-XI monoclonal antibody (hepatoma monoclonal antibody HAb18 F(ab')2) in vivo. 24 cases of primary hepatocelluar carcinoma (PHC) were equally divided into the low dose group, middle dose group and high dose group. After the relevant injection was administrated into the hepatic artery of each case, intravenous blood and urine samples were separately collected at different time for determination of the radioactive count ratio (min(-1)). The proportion of 131I-HAb18 F(ab')2 in serum of each blood sample was determined, and the radioactive count ratio (min(-1)) of druggery for each blood sample was revised according to the proportion. The pharmacokinetic parameters were calculated using DAS ver 1.0 (Drug And Statistics for Windows) program. The component of urine radiomaterial was determined and the percentages of urine radioactivity in administration dosage were calculated. The catabolism of the injection with time accorded with dynamics two-compartment model. The catabolism product was mainly free-131I and was excreted via kidney; the urine radioactivity was 47.70%-51.16% of administration dosage during 120 h after administration of drug. Therefore, the pharmacokinetics of the injection can satisfy the clinical demands. The drug dose recommended for clinical use was 27.75 MBq of the injection for each kg of human body.

Inhibition of cell proliferation and C-myc cancer protein expression in human colon adenocarcinoma cell line HT29 with VIP-131I-ASON / 生物医学工程学杂志

A 15-mer phosphorothioate antisense oligonucleotide (ASON) complementary to the translation start region of the C-myc oncogene mRNA was labeled with 131I or 125I and the labelled compound was linked to the vasoactive intestinal peptide (VIP) to be bound covalently to a polylysine chain so as to deliver oligonucleotide into tumor cells. The effect of the VIP as carrier on cell uptake of ASON in tissue culture was evaluated in a human colon adenocarcinoma HT29 cell line. The efficacy of VIP-131-ASON on cell growth was evaluated using the MTT assay. Expression of c-myc-encoded protein was measured by flow cytometry. Sense and nosense control Oligonucleotides with VIP carrier were used as control. The results showed that VIP competed effectively with VIP-125I-ASON to bind the HT29 cells. Cell uptake was increased 3-4 fold using the VIP carrier compared to the same dosage of naked DNA. HT29 cells treated with VIP-131I-ASON complexes exhibited 4-fold lower proliferation than those treated with 13I-ASON and 6-fold lower proliferation than those treated with radioiodinated Sense and nosense DNA. Cancer protein expression of HT29 cells treated with VIP-131I-ASON was decreased 2-fold compare with that in 131I-ASON treated cell. The use of a VIP carrier greatly increased 131I-ASON cellular uptake and inhibition of cell proliferation and C-myc cancer protein expressing in HT29 cell by radioiodinated antisense Oligonucleotides.

Radiobiological effect of 131I radiolabeled recombinant human epidermal growth factor on nude mice with human breast cancer / 生物医学工程学杂志

The radiobiological effect of 131I radiolabeled recombinant human epidermal growth factor (131I-rhEGF) on nude mice with human breast cancer was assessed in this study. The tissue mainly uptaking 131I-rhEGF was found by tissue distribution assay in mice. The radiation breakdown of the tissue greatly collecting 131I-rhEGF was examined by biochemical test and biopsy in nude mice with human breast cancer. The tissue distribution assay of 131I-rhEGF in mice showed that 131I-rhEGF greatly accumulated in kidney, liver, spleen and blood. The biochemical test and biopsy revealed that 131I -rhEGF injected twice (dosing once is analogous to 14.58 GBq in a person with 50 kg, once every 14 days) had an effective killing effect on tumor but had no effect of radiation breakdown on kidney, liver,spleen and blood-cell forming tissue in mice with human breast cancer. Therefore, 131I-rhEGF is a drug unharmful to normal tissues in the course of the receptor-mediated target radiotherapy for breast cancer.

Experimental research into the inhibitory effect of 131I-recombinant human epidermal growth factor on the growth of breast cancer in vivo / 生物医学工程学杂志

This experiment was designed to study the effect of 131I-recombinant human epidermal growth factor (131I-rhEGF) on the growth of tumor in nude mice loaded with human breast cancer. Bioactivity of 131I-rhEGF and uptake of 131I-rhEGF in breast cancer tissue were verified using biodistribution experiment of 131I-rhEGF in the nude mice loaded with human breast cancer. The effect of 131I-rhEGF on the growth of tumor was assessed via the growth experiment of tumor in the nude mice loaded with human breast cancer. The ultrastructural change of the tumor cell treated with 131I-rhEGF was observed under transmission electron microscope, and the pathological change of the tumor tissue treated with 131I-rhEGF was detected by biopsy. The results showed that the tumor tissue of nude mice bearing human breast cancer obviously takes in 131I-rhEGF; that intravenous administration and intratumoral administration of 131I-rhEGF both obviously inhibit the growth of tumor, the inhibition rates (82.00% and 80.70%) being remarkably higher than that of 131I (7.49%) and that of 131I-HSA (6.91%) (P<0.05); and that intravenous and intratumoral administration of 131I-rhEGF both obviously damage and kill tumor cells. Therefore, 131I-rhEGF can inhibit the growth of human breast cancer cell in nude mice; it is a potential receptor-mediated radioactivity targeting drug for treating breast cancer.

Study on permeability of brain derived neurotrophic factor through placental barrier and fetal blood brain barrier after transient uteroplacental ischemia / 中华围产医学杂志

Objective To investigate whether exogenic brain derived neurotrophic factor (BDNF) can permeate placental barrier into fetus and further through fetal blood brain barrier(BBB) after transient ischemia. Methods Seventeen day pregnant rats were selected. The uterine arteries of the rats were clamped for 30 minutes in experimental group. BDNF labeled with 125 I was injected into the rats through caudal veins. The radioactivity of BDNF in different fetal organs was measured at 24 h, 48 h and 72 h. Results 125 I BDNF was detected in amniotic fluid, placenta and fetal organs including brain, heart, lung, liver and kidney. This indicated that BDNF partly permeated placental barrier. The permeability of BDNF through placenta barrier and fetal BBB increased with the increased dose injected. BDNF reached the fetal brain through BBB under hypoxia eschemia condition. The rates of penetration of BDNF through placental barrier and BBB increased under the condition of fetal ischemia and hypoxia. Conclusions Exogenic BDNF may partly go through placental barrier and BBB into fetal brain, which makes it possible for BDNF to be a treatment for fetus suffered from ischemia and hypoxia.

Preparation of liposome-mediated 99m-technetium-labeled antisense oligonucleotides of c-myc mRNA / 生物医学工程学杂志

To explore the preparation method of liposome-mediated 99m-technetium-labeled antisense oligonucleotides of c-myc mRNA and lay foundations for antisense imaging and treatment, antisense oligonucleotides (DNA) with 15 bases and di-functional chelate, hydrazino nicotinamide derivatives, were synthesized. After DNA combined with chelate, they were labeled with 99m-technetium to form compounds, 99mTc-chelate-DNA (99mTc-DNA), and were purified through Sep-Pak reverse column(C18, Waters) by using methanol and water as eluent. The leaching curve was made; the labeling efficiency was calculated. The products were then encapsulated with cation liposome to form liposome-mediated 99mTc-DNA. The radiochemical purity and stability of the liposome-mediated 99mTc-DNA were tested through strip chromatography. The labeling efficiency was 63.37% +/- 3.51% at the radioactive concentration of 1480 MBq, 62.52% +/- 3.69% at that of 740 MBq, 59.82% +/- 5.12% at that of 592 MBq. There were no significant differences between these labeling efficiencies. The radiochemical purity was 96.47% +/- 3.01%. The liposome-mediated radiolabeled antisense oligonucleotides were stable after incubation with water or serum. Therefore liposome-mediated radiolabeled antisense oligonucleotides could be obtained through hydrazino nicotinamide derivatives as di-functional chelate and liposome as vector.

The primary applications of ¹⁵³Sm-EDTMP plus chemotherapy in the treatment of bone metastasis of lung cancer / 中国肺癌杂志

<p><b>BACKGROUND</b>To study the clinical effects of ¹⁵³Sm-EDTMP plus chemotherapy in the treatment of bone metastasis of lung cancer.</p><p><b>METHODS</b>One hundred and ten lung cancer patients with one metastasis [male 82 and female 28, aged from 32 to 76 yrs; squamous cell carcinoma 28, adenocarcinoma 27, small cell lung cancer (SCLC) 7, mix type 41, alveolar carcinoma 7] who did not undergo an operation were entered into this study. The patients were divided into 3 groups: ¹⁵³Sm-EDTMP therapy only (37 cases), ¹⁵³Sm-EDTMP plus chemotherapy after 3 days (42 cases), 30 days after chemotherapy plus ¹⁵³Sm-EDTMP (31 cases). The dosages of ¹⁵³Sm-EDTMP ranged from 1 111 to 2 660 MBq. The patients with SCLC were adapted CCNU, MTX and CTX; those with non-small cell lung cancer (NSCLC) were adapted MMC, VCR and DDP. Statistic analysis of the data was performed by Chi-square test.</p><p><b>RESULTS</b>Total pain relief rate for ¹⁵³Sm-EDTMP only was 89.2% , for ¹⁵³Sm-EDTMP plus chemotherapy was 92.8%, and for chemotherapy plus 153 Sm EDTMP was 90.3% . The foci disappeared in 9 cases with ¹⁵³Sm-EDTMP only, in 12 cases with ¹⁵³Sm-EDTMP plus chemotherapy, and in 9 cases with chemotherapy plus ¹⁵³Sm-EDTMP. The 1 year survival rate was 29.7%(11/37) by 153 Sm only, 40.5%(17/42) by 153 Sm plus chemotherapy, 38.7%(12/31) by chemotherapy plus ¹⁵³Sm-EDTMP.</p><p><b>CONCLUSIONS</b>¹⁵³Sm-EDTMP plus chemotherapy is effective in the treatment of bone metastasis of lung cancer.</p>