[Stable expression of human thyrotropin receptor in HEK 293T cells].

OBJECTIVE: To establish a strain of eukaryotic cells with overexpressed human thyrotropin receptor (hTSHR) for the development of anti-thyroid drugs. METHODS: GV266 vector and hTSHR gene were digested by Age I/Nhe I. Plasmids expressing GV266-hTSHR were constructed using T4 ligase and then transfected into 293T cells. The expression of hTSHR was determined by Western blot. Packaging plasmids were built in the 293T cells with Opti-MEM, Lipofectamine 2000 and helper plasmid. The titer of the packaging plasmids was determined with qPCR. The packaging plasmids and the plasmids expressing GV266-hTSHR were co-transfected into 293T cells to obtain a strain of cells (GV266-hTSHR-293T) with stable expression of hTSHR. The GV266-hTSHR293T stain was detected by green fluorescent protein (GFP) fluorescence. RESULTS: The DNA sequence of GV266-hTSHR matched that of hTSHR. The Western blot showed a 62 x 10(3) target band. The titer of packaging plasmids reached 2 x10(8) TU/mL. The GV266-hTSHR-293T cells were visible under GFP fluorescence. CONCLUSION: HEK 293T cells with stable expression of hTSHR was established.

[TgAb interference with experimental Tg values detected by dilution curves].

OBJECTIVE: To identify the interference of endogenous antithyroglobulin (TgAb) with experimental thyroglobulin (Tg) values using dilution curves. METHODS: Dilution buffer, detectable TgAb serums (TgAb 20-25 IU/mL, Tg free) and undetectable TgAb serums (TgAb < 10 IU/mL, Tg free) were employed to dilute the Tg serums from patients with DTC, respectively. The Tg values were detected by immunometric assay (IMA). The experimental Tg values (Y-axis) were plotted against expected serum Tg values (X-axis). Diluted curves were used to evaluate the interference of TgAb on the experimental Tg values. A linear dilution curve is supposed to appear if no TgAb interference exists. RESULTS: The Tg dilution curves with dilution buffer were linear. Thirty six dilution curves were obtained with TgAb serums from six patients diluted by detectable TgAb serums, and 12 showed linear. Tg serums from six patients diluted by one detectable TgAb serum resulted in both linear and non-linear results. One Tg serum diluted by six TgAb serums also resulted both linear and non-linear results. Tg serums from three patients diluted by five undetectable TgAb serums resulted in 11 dilution curves, four of which were linear. CONCLUSION: Dilution curves can be used to predict TgAb interference indirectly. Detectable TgAb may not interfere with experimental Tg values. Whereas, undetectable TgAb may interfere with Tg values. TgAb could not be used to predict Tg interference.

[The correlation between endogenous TgAb and experimental Tg values in patients with differentiated thyroid carcinomas].

OBJECTIVE: To determine the interference of antithyroglobulin (TgAb) on the measurement of serum thyroglobulin (Tg) in patients with differentiated thyroid carcinomas (DTC). METHODS: The Tg positive serum and TgAb positive serum were taken from the patients with total thyroidectomy and thyroid remnant ablation by radioiodine. The Tg positive serum contained high level of Tg (>10 microg/L) and nondetectable TgAb. The TgAb positive serum contained high level of TgAb (>115 IU/mL) and nondetectable Tg. We incubated a constant amount of Tg with increasing volumes of Tg-free autoantibodies and allowed them to equilibrate at room temperature for 12 hours before measuring Tg and TgAb. The thyroglobulin and TgAb values were determined by the Roche electrode induce chemiluminescent emission system with monoclonal antibody sandwich analysis. The correlation between TgAb and serum Tg values was analysed. RESULTS: TgAb led to underestimation of serum Tg values by 15%-50% in 8 patients with DTC. TgAb was correlated with the magnitude of underestimated serum Tg values in 3 patients with DTC, but not in the other 5 patients with DTC. CONCLUSION: TgAb underestimated serum Tg values in a dose-dependent manner in some patients with DTC, but not in others.

Technetium-99m-labeled macroaggregated albumin lung perfusion scan for diagnosis of hepatopulmonary syndrome: A prospective study comparing brain uptake and whole-body uptake.

BACKGROUND: Hepatopulmonary syndrome (HPS) is an arterial oxygenation defect induced by intrapulmonary vascular dilatation (IPVD) in the setting of liver disease and/or portal hypertension. This syndrome occurs most often in cirrhotic patients (4%-32%) and has been shown to be detrimental to functional status, quality of life, and survival. The diagnosis of HPS in the setting of liver disease and/or portal hypertension requires the demonstration of IPVD (i.e., diffuse or localized abnormally dilated pulmonary capillaries and pulmonary and pleural arteriovenous communications) and arterial oxygenation defects, preferably by contrast-enhanced echocardiography and measurement of the alveolar-arterial oxygen gradient, respectively. AIM: To compare brain and whole-body uptake of technetium for diagnosing HPS. METHODS: Sixty-nine patients with chronic liver disease and/or portal hypertension were prospectively included. Brain uptake and whole-body uptake were calculated using the geometric mean of technetium counts in the brain and lungs and in the entire body and lungs, respectively. RESULTS: Thirty-two (46%) patients had IPVD as detected by contrast-enhanced echocardiography. The demographics and clinical characteristics of the patients with and without IPVD were not significantly different with the exception of the creatinine level (0.71 ± 0.18 mg/dL vs 0.83 ± 0.23 mg/dL; P = 0.041), alveolar-arterial oxygen gradient (23.2 ± 13.3 mmHg vs 16.4 ± 14.1 mmHg; P = 0.043), and arterial partial pressure of oxygen (81.0 ± 12.1 mmHg vs 90.1 ± 12.8 mmHg; P = 0.004). Whole-body uptake was significantly higher in patients with IPVD than in patients without IPVD (48.0% ± 6.1% vs 40.1% ± 8.1%; P = 0.001). The area under the curve of whole-body uptake for detecting IPVD was significantly higher than that of brain uptake (0.75 vs 0.54; P = 0.025). The optimal cut-off values of brain uptake and whole-body uptake for detecting IPVD were 5.7% and 42.5%, respectively, based on Youden's index. The sensitivity, specificity, and accuracy of brain uptake > 5.7% and whole-body uptake > 42.5% for detecting IPVD were 23%, 89%, and 59% and 100%, 52%, and 74%, respectively. CONCLUSION: Whole-body uptake is superior to brain uptake for diagnosing HPS.

[The effects of antisense oligonucleotides bcl-2/bcl-xl and bcl-2 on proliferation and apoptosis of breast cancer cells].

OBJECTIVE: To evaluate the effects of phosphorothioate antisense oligonucleotides (ASON) bcl-2/ bc-xl ASON and bcl-2 on the proliferation and apoptosis of breast cancer cells, MCF-7. METHODS: 1) bcl-2 ASON and bcl-2/bcl-xl ASON were transfected into MCF-7 cells with anionic long circulating liposomes (NA), cationic LCL (PA), respectively. 2) After incubation with bcl-2 ASON (FB1), bcl-2/bcl-xl ASON (FB2), NA loaded with bcl-2 ASON (NA-S) or bcl-2/bcl-xl ASON(NA-D), PA loaded with bcl-2 ASON(PA-S) or bcl-2/bc-xl ASON (PA-D) for 24 h, their inhibition on MCF-7 cells were evaluated by using HE staining, methythiazolyltetrazolium (MTT) and flow cytometry (FCM). RESULTS: The significant difference of nuclear condensation, chromatin fragmentation and apoptotic bodies in MCF-7 cells, typical of apoptotic cell death was observed in groups of bcl-2/ bcl-xl bispecific ASON by compared with that of bcl-2 ASON treatment. The fluorensence intensities of bcl-2 in groups of NA-D and NA-S, PA-S and PA-D, FB1 and FB2 were 1.92+/-0.08 and 2.83+/-0.16 (P=0.028); 4.20+/-0.18 and 2.85+/-0.57 (P=0.001); 5.70+/-1.16 and 4.35+/-0.11 (P=0.001), respectively. The cell survival rates at 24 h of NA-D and NA-S, PA-S and PA-D, FB1 and FB2 were (0.32+/-0.03)% and (0.58+/-0.07)% (P=0.014); (0.71+/-0.03)% and (0.45+/-0.04)% (P=0.014); (0.88+/-0.04)% and (0.57+/- 0.05)% (P=0.003), respectively. CONCLUSION: The bcl-2/bct-xl bispecific ASON could inhibit bcl-2 expression and induce apoptosis of breast cancer cells more efficiently than that treated with bcl-2 ASON alone.

[Preparation of 125I-oligonucleotide-long-circulation liposomes].

OBJECTIVE: To investigate the preparation and the encapsulation efficiency of 125I-oligonucleotide(ON)-long-circulation liposomes (LCL). METHODS: (1) Oligonucleotide was labeled with 125I using thallium chloride tetrahydrate (TICl3) as an oxidizing agent. 125I-oligonucleotide was separated from free 125I by Sephadex G25 column chromatography. The following radiolabeling efficiency, radiochemistry purity and specific radioactivity were obtained respectively. Subsequently, the stability of 125I-ON was observed. (2) 125I-ON-LCL were prepared by means of reverse-phase evaporation. The quality of 125I-ON-LCL was evaluated after the crude LCL were repeatedly extruded through 400 nm, 200 nm, 100 nm polycarbonate membranes consecutively. Results (1) The radiolabeling efficiency, radiochemistry purity and specific radioactivity of 125I-antisense oigonucleotide (ASON) were (72.80 +/- 0.68)%, (98. 33 +/- 0.39)% and (0.63 +/- 0.11) MBq/microg; of 125I-sense oigonucleotide (SON) were (72.21 +/- 0.60)%, (98.28 +/- 0.36)%, (0.63 +/- 0.14) MBq/microg; and of 125I-nonsense oligonucleotide (NON) were (72.77 +/- 0.81)%, (98.42 +/- 0.40)%, (0.62 +/- 0.11) MBq/microg, respectively. (2) The radiochemistry purity of 125I-ON, in 0.01 mol/L HEPES buffer and human serum at 1 h, 2 h and 4 h were all above 93%, 80%, respectively. (3) LCL formulations were 115 nm in mean diameter with a PDI (polydispersibility index) of 0.103 and Zeta potential of -29.23. The encapsulation efficiencies of 125I-ASON, 125I-SON and 125I-NON were (66.21 +/- 0.21)%, (70.93 +/- 0.03)% and (67.67 +/- 0.10)% respectively. CONCLUSION: LCL were prepared in high loading efficiency for 125I-ON with small particle sizes and symmetric distributions.