[Research on the knockout of LMNA gene by CRISPR/Cas9 system in human cell lines].

The LMNA gene encodes the nuclear Lamin A and Lamin C proteins, and is related to nuclear membrane organization, genome stability and cell differentiation. Abnormal expression of LMNA is ubiquitous in human tumors, and its mutation leads to various forms of laminopathies, including Emery-Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy (DCM), and Hutchinson-Gliford progeria syndrome (HGPS). To further determine the functions of the LMNA gene in cellular physiology, the present study used the CRISPR/Cas9 technique to edit the LMNA gene of 293T and HepG2 cells in vitro, which resulted in two stable LMNA gene knockout (LMNA KO) cell lines. Compared to the respective wild type cells, the LMNA KO cell lines showed decrease in proliferation ability, increase in apoptosis, alteration in cellular morphology and uneven structures in the nucleus membrane. In this study, we report for the first time the results on the construction of LMNA KO immortalized cell lines and characterization of their morphological changes, thereby laying the foundation for the further studies of the LMNA gene functions and pathogenic mutations.

Low-density lipoprotein level on admission is not associated with postintravenous thrombolysis intracranial hemorrhage in patients with acute ischemic stroke.

Intravenous thrombolysis with the tissue plasminogen activator (tPA) is the gold standard for acute ischemic stroke. However, its application is limited because of the concern of the post-tPA intracranial hemorrhage (ICH). Low low-density lipoprotein (LDL) has been speculated to increase the risk of hemorrhagic transformation after ischemic stroke. However, whether LDL is associated with post-tPA ICH remains controversial. The present study obtained the medical records from Shuang Ho Hospital and retrospectively reviewed for the period between August 2009 and December 2016 to investigate the association between LDL and the risk of post-tPA ICH. The differences were analyzed using the Student's t-test, Fisher's exact test, the univariate and stepwise multiple regression model, and p<0.05 was considered statistically significant. Among 218 patients, post-tPA ICH was noted in 23 (10.5%) patients. Patients with post-tPA ICH tended to have a lower LDL level (ICH group: 102.00±24.56, non-ICH group: 117.02±37.60 mg/dL, p=0.063). However, after adjustment for the factors might affect the risk of post-tPA ICH, such as stroke severity, onset-to-treatment time interval, and atrial fibrillation (AF), LDL level was not associated with post-tPA ICH whereas AF was the only significant factor increased the risk of post-tPA ICH (adjusted OR: 1.177, 95% CI 1.080 to 1.283). In addition, patients with AF had significant lower LDL level and for patients without AF, LDL was not associated with the post-tPA ICH. In conclusion, LDL level is not associated with the risk of post-tPA ICH in Taiwanese patients with stroke.

[Synthesis of polyrotaxane-camptothecin conjugates and evaluation of its anti-tumor effect].

To prepare polyrotaxane-camptothecin conjugates and evaluate its anti-tumor effect, polyrotaxane-camptothecin conjugates were successfully synthesized, and the release behavior was performed; MTT assay and cell morphology were used to examine the inhibition of cells' proliferation effect in vitro. The experimental study of the antitumor effect on S180 mice in vivo was also performed to further evaluate the anti-tumor effect of conjugate. The result showed polyrotaxane-camptothecin conjugates can effectively inhibit the proliferation in a dose dependent effect. In vivo study and cell morphology observation of S180 mice showed significant decrease in growth of tumor, degree of tumor infiltration and blood vessel number. The result indicated anti-tumor mechanism may be through affect the angiogenesis and reduced blood supply to tumor cells and then leading to necrosis.

[Establishment of microarray for detecting mutation in HBV pre-core/core and basic core promoter regions].

OBJECTIVE: To develop a sensitive and specific microarray for detecting mutations of HBV pre-core/core and basic core promoter regions in the clinic. METHODS: Site-specific oligonucleotide probes were designed and immobilized to microarray slides and hybridized to HBV gene fragments amplified with specific biotin-labeled primer using asymmetrical PCR. The specificity and sensitivity of the method were estimated. And the microarray was applied to detect 138 clinical serum samples with HBV-DNA. RESULTS: The mutations of HBV pre-core/core and basic core promoter regions can be specifically detected using the microarray, and the sensitivity was 1 x 10(1) copies/microl. Among 138 samples, 40 samples had T1762/ A1764 mutation, 11 samples had C1814 mutation, and 16 samples had A1896 mutation. The A1896 mutation rate in high HBV-DNA load group was significantly higher than that in low HBV-DNA load group (P < 0.01). CONCLUSION: An DNA microarray assay was successfully established to detect the mutations in HBV pre-core/core and basic core promoter regions. The A1896 mutation in pre-core/core region maybe involve in duplication of HBV.

[Preparation of Ginkgo biloba extract 50-phospholipid complex and study on its physicochemical properties].

OBJECTIVE: To optimize the preparation technology of GBE 50-phospholipid complex and study its physicochemical properties. METHODS: The preparation conditions for GBE 50-phospholipid complex were optimized by means of single factor study and orthogonal design, and taking the complexing rate of total flavonoids as assessment criteria, the complex was analyzed by DSC, IR and determined apparent oil-water distribution coefficients in different pH aqueous solution. RESULTS: The optimized preparation conditions for GBE 50-phospholipid complex were obtained as follows: the solvent was Tetrahydrofuran, the temperature was 30 degrees C, the concentration of GBE 50 was 20 mg/mL, the ratio of GBE 50 to phospholipids was 1 to 1, and the complexing rate was 98%. The complex significantly improved GBE 50 on the solubility in octanol, also on the oil-water apparent partition coefficient. CONCLUSION: GBE 50-phospholipid complex is very different from GBE 50 on the physicochemical properties.

[Preparation of the oral self-microemulsifying drug delivery system of GBE50].

To prepare the oral self-microemulsifying drug delivery system (SMEDDS) of GBE50, balance solubility method was used to screen emulsifier and assistant emulsifier; a pseudo-tamary phase diagram was used to prepare microemulsion; and orthogonal design was used to optimize formulation. Self-microemulsifying efficiency, dissolution, stability and pharmacokinetics of the preparation were studied. As a result, GBE50-SMEDDS of IPM, Cremophor EL, 1,2-propanediol and GBE50 could be self emulsified to form stable microemulsion with particle diameter between 20 and 50 nm when emulsifying with water. Its self-microemulsifying efficiency and dissolution are quick with good stability and it has a higher bioavailability than market existing agents Xingling particles. GBE50-SMEDDS is stable and effective.