Application of percutaneous pie-crusting deep medial collateral ligament release for posterior horn surgery of medial meniscus / 中国骨伤

OBJECTIVE@#To explore clinical and radiographic effects of percutaneous pie-crusting deep medial collateral ligament release in patients with posterior horn tear of medial meniscus combined with tight medial compartment.@*METHODS@#From January 2012 to December 2016, 35 patients with medial meniscus posterior horn injury were treated with percutaneous pie crusting deep medial collateral ligament release technique, including 21 males and 14 females, aged from 21 to 55 years old with an average of (39.1±6.5) years old. Degree of meniscus extrusion were recorded before and 24 months after operation. The knee valgus stress test was performed to evaluate stability of medial collateral ligament, and compared difference between healthy and affected side. Lysholm and IKDC functional scores were compared before and 24 months after operation.@*RESULTS@#All patients were followed up from 27 to 60 months with an average of (36.7±6.8) months. All patients were underwent operation, the wound healed well without complications. Operative time ranged from 0.5 to 1.2 h with an average of (0.8±0.4) h. Nineteen patients were performed partial meniscectomy, 16 patients were performed repair suture. Convex of meniscus before operation was (1.5±0.7) mm, and (1.7±0.4) mm after operation;had no statistical difference(@*CONCLUSION@#For patients with medial meniscus tear of posterior horn combined with tight medial compartment, percutaneous pie-crusting deep medial collateralligament release could improve medial compartment space, and Knee valgus instability and meniscus extrusion are not affected.

Enhancement of artemisinin biosynthesis in transgenic Artemisia annua L. by overexpressed HDR and ADS genes / 药学学报

Artemisnin is a novel sesquiterpene lactone with an internal peroxide bridge structure, which is extracted from traditional Chinese herb Artemisia annua L. (Qinghao). Recommended by World Health Organization, artemisinin is the first-line drug in the treatment of encephalic and chloroquine-resistant malaria. In the present study, transgenic A. annua plants were developed by overexpressing the key enzymes involved in the biosynthetic pathway of artemisinin. Based on Agrobacterium-mediated transformation methods, transgenic plants of A. annua with overexpression of both HDR and ADS were obtained through hygromycin screening. The genomic PCR analysis confirmed six transgenic lines in which both HDR and ADS were integrated into genome. The gene expression analysis given by real-time quantitative PCR showed that all the transgenic lines had higher expression levels of HDR and ADS than the non-transgenic control (except ah3 in which the expression level of ADS showed no significant difference compared with control); and the HPLC analysis of artemisinin demonstrated that transgenic A. annua plants produced artemisinin at significantly higher level than non-transgenic plants. Especially, the highest content of artemisinin was found in transgenic line ah70, in which the artemisinin content was 3.48 times compared with that in non-transgenic lines. In summary, overexpression of HDR and ADS facilitated artemisinin biosynthesis and this method could be applied to develop transgenic plants of A. annua with higher yield of artemisinin.

Expression pattern of genes involved in tropane alkaloids biosynthesis and tropane alkaloids accumulation in Atropa belladonna / 中国中药杂志

Atropa belladonna is a medicinal plant and main commercial source of tropane alkaloids (TAs) including scopolamine and hyoscyamine, which are anticholine drugs widely used clinically. Based on the high throughput transcriptome sequencing results, the digital expression patterns of UniGenes representing 9 structural genes (ODC, ADC, AIH, CPA, SPDS, PMT, CYP80F1, H6H, TRII) involved in TAs biosynthesis were constructed, and simultaneously expression analysis of 4 released genes in NCBI (PMT, CYP80F1, H6H, TRII) for verification was performed using qPCR, as well as the TAs contents detection in 8 different tissues. Digital expression patterns results suggested that the 4 genes including ODC, ADC, AIH and CPA involved in the upstream pathway of TAs, and the 2 branch pathway genes including SPDS and TRII were found to be expressed in all the detected tissues with high expression level in secondary root. While the 3 TAs-pathway-specific genes including PMT, CYP80F1, H6H were only expressed in secondary roots and primary roots, mainly in secondary roots. The qPCR detection results of PMT, CYP80F1 and H6H were consistent with the digital expression patterns, but their expression levels in primary root were too low to be detected. The highest content of hyoscyamine was found in tender stems (3.364 mg x g(-1)), followed by tender leaves (1.526 mg x g(-1)), roots (1.598 mg x g(-1)), young fruits (1.271 mg x g(-1)) and fruit sepals (1.413 mg x g(-1)). The highest content of scopolamine was detected in fruit sepals (1.003 mg x g(-1)), then followed by tender stems (0.600 mg x g(-1)) and tender leaves (0.601 mg x g(-1)). Both old stems and old leaves had the lowest content of hyoscyamine and scopolamine. The gene expression profile and TAs accumulation indicated that TAs in Atropa belladonna were mainly biosynthesized in secondary root, and then transported and deposited in tender aerial parts. Screening Atropa belladonna secondary root transcriptome database will facilitate unveiling the unknown enzymatic reactions and the mechanisms of transcriptional control.

Enhancement of tropane alkaloids production in transgenic hair roots of Atropa belladonna by overexpressing endogenous genes AbPMT and AbH6H / 药学学报

Atropa belladonna L. is the officially medicinal plant species and the main commercial source of scopolamine and hyoscyamine in China. In this study, we reported the simultaneous overexpression of two functional genes involved in biosynthesis of scopolamine, which respectively encoded the upstream key enzyme putrescine N-methyltransferase (PMT; EC 2.1.1.53) and the downstream key enzyme hyoscyamine 6beta-hydroxylase (H6H; EC 1.14.11.11) in transgenic hair root cultures of Atropa belladonna L. HPLC results suggested that four transgenic hair root lines produced higher content of scopolamine at different levels compared with nontransgenic hair root cultures. And scopolamine content increased to 8.2 fold in transgenic line PH2 compared with that of control line; and the other four transgenic lines showed an increase of scopolamine compared with the control. Two of the transgenic hair root lines produced higher levels of tropane alkaloids, and the content increased to 2.7 fold in transgenic line PH2 compared with the control. The gene expression profile indicated that both PMT and H6H expressed at a different levels in different transgenic hair root lines, which would be helpful for biosynthesis of scopolamine. Our studies suggested that overexpression of A. belladonna endogenous genes PMT and H6H could enhance tropane alkaloid biosynthesis.