Tissue Extracts From Infarcted Myocardium of Rats in Promoting the Differentiation of Bone Marrow Stromal Cells Into Cardiomyocyte-like Cells

Objective To investigate whether cardiac tissue extracts from rats could mimic the cardiac microenvironment and act as a natural inducer in promoting the differentiation of bone marrow stromal cells （BMSCs） into cardiomyocytes. Methods Three kinds of tissue extract or cell lysate [infarcted myocardial tissue extract （IMTE）, normal myocardial tissue extract （NMTE） and cultured neonatal myocardial lysate （NML）] were employed to induce BMSCs into cardiomyocyte-like cells. The cells were harvested at each time point for reverse transcription-polymerase chain reaction （RT-PCR） detection, immunocytochemical analysis, and transmission electron microscopy. Results After a 7-day induction, BMSCs were enlarged and polygonal in morphology. Myofilaments, striated sarcomeres, Z-lines, and more mitochondia were observed under transmission electron microscope. Elevated expression levels of cardiac-specific genes and proteins were also confirmed by RT-PCR and immunocytochemistry. Moreover, IMTE showed a greater capacity of differentiating BMSCs into cardiomyocyte-like cells. Conclusions Cardiac tissue extracts, especially IMTE, can effectively differentiate BMSCs into cardiomyocyte-like cells.

DETECTION AND CLINICAL SIGNIFICANCE OF THROMBOMODULIN IN BOTH PLASMA AND TISSUE EXTRACTS OF CANCER PATIENTS

To study the changes of thrombomodulin(TM) in both plasma and tissue extracts of cancer patients for evaluating its clinical significance. Methods: PlasmaTM levels were measured by enzyme-linked immunosorbent assay (ELISA) in both plasma of 188 cancer patients and 24 cancer tissue extractsincluding their adjacent non-cancer tissues. Results:The plasma TM levels both in cancer patients and in metastasis patients were significantly higher than that in controls [(33.4714.25)mg/L, (41.6816.96)mg/L, vs(20.40 7.22) mg/L,P<0.01]. The plasma TM levels incancer patients after operation decreased obviously thanthat before operation [(18.459.96)mg/L, vs (28.2911.74)mg/L, P<0.01], whereas, the plasma TM levels in patientswith recurrence and metastasis after operation increasedobviously [(34.5012.57)mg/L]. Among the types of cancer,the plasma TM levels in metastasis lung cancers, gastric cancers and pancreatic cancers were significantly higherthan that in non-metastasis respective cancers. Nosignificant differences were found between controls andnon-metastasis cancers including gastric cancers,pancreatic cancers, nasopharyngeal cancers, large intestine cancers and laryngeal cancers (P>0.05). The TM levels incancer tissue extracts were significantly lower than that intheir adjacent non-cancer tissue extracts [(647.71317.51)mg/L vs (1455.63772.22)mg/L, P<0.01]. On the contrary, the plasma TM levels in these cancers were significantly higher than that in controls. Conclusion: The rise of plasma TMlevels in cancer patients was associated with metastasis and diffusion of cancers. The TM levels can be served as ansensitive index for judging progression and metastasis of

Effects of regenerated tissue extracts after liver injury on the proliferation,differentiation,migration and invasion of SK-HEP1 cells

Objective: To study the effects of regenerated tissue extracts after liver injury on the proliferation, differentiation, migration and invasion of SK-HEP1 cells. Methods: Regenerated tissue extracts after liver injury were used to induce SK-HEP1 cells after enrichment, their effects on the proliferation, differentiation, migration and invasion of SK-HEPI cells were observed through in vitro cell culture, MTT, flow cytometry and transwell assays. Results:In response to the action of regenerated tissue extracts after liver injury, SK-HEP1 cells were blocked in G_0/G_1 phase, their growth rate was distinctly reduced. The number of SK-HEP1^(-fj)colonies decreased. The migration ability of SK-HEPI cells showed a decreased trend on day7 and day 11 after induction. SK-HEPl's invasion ability clearly decreased on days 7 and11 after induction, especially on day 7. Conclusions: To a certain extent, regenerated tissue extracts after liver injury can inhibit the proliferation, differentiation, migration and invasion of hepatoma cells, showing an important potential of being a differentiating agent for the treatment of liver cancer.

Smashing Tissue Extraction of Flavonoids in Lophatherum gracileSmashing Tissue Extraction of Flavonoids in Lophatherum gracile

Objective] The study aimed to optimize the procedures for flavonoids ex-traction from of Lophatherum gracile. [Method] The powder of L. gracile leaf （3.0 g） was weighed and extracted fol owing an orthogonal design including the solid-liquid ratio, extraction time, extraction times and soaking time at three levels. [Result] The optimal parameters were solid-liquid ratio at 1：40, extraction time of 40 s, extraction times of two times and soaking time of 40 min. [Conclusion] Smashing tissue ex-traction of flavonoids is rapid and efficient, which provides a new method for the development and utilization of L. gracile.

Novel distribution pattern of fibrinolytic components in rabbit tissues extract: a preliminary study

Objective: The purpose of this work was to investigate the distribution pattern of fibrinolytic factors and their inhibitors in rabbit tissues. Methods: The components of the fibrinolytic system in extracts from a variety of rabbit tissues, including tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), plasminogen (Plg), plasmin (Pl) and α2 plasmin inhibitor (α2PI), were determined by colorimetric assay. Results: The tissue extracts in renal, small intestine, lung, brain and spleen demonstrated strong fibrinolytic function, in which high activity of tPA, Plg and Pl was manifested; whereas in skeletal muscle, tongue and stomach, higher activity of PAI-1 and α2PI showed obviously. Also excellent linear correlations were found between levels of tPA and PAI-1, Pl and α2PI, Plg and Pl. In related tissues, renal cortex and renal marrow showed distinctly higher activity of tPA and lower activity of PAI-1, with the levels of Plg and Pl in renal cortex being higher than those in renal marrow, where the α2PI level was higher than that in renal cortex. Similarly, the levels of tPA, Plg and Pl in small intestine were higher than those in large intestine, but with respect to PAI-1 and α2PI, the matter was reverse. In addition, the fibrinolytic activity in muscle tissue was lower, however, the levels of tPA, Plg, and Pl in cardiac muscle were obviously higher than those in skeletal muscles, and the levels of PAI-1 and α2PI were significantly lower than those in skeletal muscle. Conclusion: Our data demonstrate that a remarkable difference of the fibrinolytic patterns exists in rabbit tissues, which has probable profound significance in understanding the relationship between the function of haemostasis or thrombosis and the physiologic function in tissues.

Progesterone promotes neuronal differentiation of human umbilical cord mesenchymal stem cells in culture conditions that mimic the brain microenvironment

In this study, human umbilical cord mesenchymal stem cells from full-term neonates born by vagina delivery were cultured in medium containing 150 mg/mL of brain tissue extracts from Sprague-Dawley rats （to mimic the brain microenvironment）. Immunocytochemical analysis demonstrated that the cells differentiated into neuron-like cells. To evaluate the effects of progesterone as a neurosteroid on the neuronal differentiation of human umbilical cord mesenchymal stem cells, we cultured the cells in medium containing progesterone （0.1, 1, 10 pM） in addition to brain tissue extracts. Reverse transcription-PCR and flow cytometric analysis of neuron specific enolase-positive cells revealed that the percentages of these cells increased significantly following progesterone treatment, with the optimal progesterone concentration for neuron-like differentiation being 1 tJM. These results suggest that progesterone can enhance the neuronal differentiation of human umbilical cord mesenchymal stem cells in culture medium containing brain tissue extracts to mimic the brain microenvironment.

Biochemical effects of gadolinium chloride in rats liver and kidney studied by ^1H NMR metabolomics

The biochemical effects of gadolinium chloride were studied using high-resolution IH nuclear magnetic resonance （NMR） spec- troscopy to investigate the biochemical composition of tissue （liver and kidney） aqueous extracts obtained from control and gadolinium chloride （GdCl3） （10 and 50 mg/kg body weight, intraperitoneal injection, i.p.） treated rats. Tissue samples were collected at 48, 96 and 168 h p.d. after exposure to GdCI3, and extracted using methanol/chloroform solvent system. ^1H NMR spectra of tissue extracts were analyzed by pat- tern recognition using principal components analysis. The liver damages caused by GdCl3 were characterized by increased succinate and decreased glycogen level and elevated lactate, alanine and betaine concentration in liver. Furthermore, the increase of creatine and lactate, and decrease of glutamate, alanine, phosphocholine, glycophosphocholine （GPC）, betaine, myo-inositol and trimethylamine N-oxide （TMAO） levels in kidney illustrated kidney disturbance induced by GdCl3.

Smashing Tissue Extraction and HPLC Determination of Active Saponins from Different Parts of Panax notoginseng

Objective To optimize the extraction technology used for extracting active saponins from the roots,fibrous roots,basal part of stems,root verrucae,fruits,flowers,stems,and leaves of Panax notoginseng based on the contents of ginsengsides Rg1,Rb1,and notoginsengside R1 as evaluation indexes.Methods Different parts of P.notoginseng were extracted by smashing tissue extraction(STE),ultrasound extraction,and reflux extraction.The contents of ginsengsides Rg1,Rb1,and notoginsengside R1 in 24 kinds of extracts were determined by HPLC-UV.Hypersil C18 column(200 mm × 4.6 mm,5 μm) and acetonitrile-warter(20:80 for 30 min→45:55 for 18 min→70:30 for 2 min→80:20 for 10 min→100:0) were used;UV detector was set at 203 nm;The flow rate was set at 1.0 mL/min.Results STE was the most efficient technology with the highest yield of active saponins among the three tested extraction technologies.Conclusion STE is a fast,effective,and economical method to extract the active saponins from different parts of P.notoginseng.It could significantly shorten the extraction time and simplify the determination of the pre-processing work on identifying P.notoginseng.Such quick and effective extraction provides a powerful tool for analyzing P.notoginseng in the future.

Smashing Tissue Extraction and GC Analysis of Active Fatty Acids from Oil Cake of Perilla Seeds

Objective To optimize the extraction technology of perilla seeds oil from the oil cake of perilla seeds(OCPS)by using the contents of active fatty acids as evaluation standard.Methods The fatty acids were extracted from OCPS,the residue of perilla seeds after cold-press,by smashing tissue extraction(STE),the new technology selected through comparing with classical leaching extraction(LE),Soxhlet extraction(SE),ultrasonic extraction(UE),and supercritical-CO2 fluid extraction(SFE).For optimized condition of STE,orthogonal test was designed and completed.The contents of five fatty acids in extracted oil and OCPS were determined by GC.Results The optimized extraction parameters were smashing for 1.5 min under extraction power of 150 W and 1:6 of the material/solvent ratio.The contents of five fatty acids in the oils extracted by five techniques from OCPS and determined by GC were as follows:α-linolenic acid(41.12%-51.81%),linoleic acid(15.38%-16.43%),oleic acid (18.93%-27.28%),stearic acid(2.56%-4.01%),and palmitic acid(7.38%-10.77%).Conclusion The results show that STE is the most efficient technology with the highest yield(LE:0.57%;SE:1.03%;UE:0.61%;SFE:0.80%;STE: 1.17%)and shortest time(LE:720 min;SE:360 min;UE:30 min;SFE:120 min;STE:1.5 min)among five tested extraction technologies.It is first reported using STE to extract herbal oil enriched with active fatty acids.

Optimization of Smashing Tissue Extraction Technology of Schisandra chinensis Fruits by Orthogonal Test

Objective To optimize the extract technology of active lignins from the fruits of Schisandra chinensis. Methods The content of schizandrin, gomisin A, and deoxyschizandrin were selected as standards to evaluate the efficiency of smashing tissue extraction (STE). Solid-liquid ratio, extracting times, ethanol concentration, and extracting time were investigated through orthogonal test. Results The optimized conditions for STE were ten times amount of 80% EtOH, extracting for three times, and 2 min for each time. Conclusion STE could obtain relatively higher yield, simplicity of operation, and benefit for environment protection. It could be better choice for the extraction of S. chinensis.

Smashing Tissue Extraction and HPLC Determination of Paclitaxel and 10-Deacetylbaccatin from Taxus x media

Objective To optimize the extraction technology of Taxus x media by using the contents of Paclitaxel and 10-deacetylbaccatin(10-DAB) ,two representative active diterpene alkaloids of taxane type from T.x media,as evaluation standard.Methods The smashing tissue extraction(STE) of Paclitaxel and 10-DAB from T.x media,was investigated by comparing with ultrasonic extraction(UE) which was one of the modern technologies of extraction.Results STE was more efficient than UE,and the contents of 10-DAB and Paclitaxel in the extracts obtained by STE were higher than those by UE.Conclusion STE is a fast,high-performance,and energy-saving technology for the extraction of diterpene alkaloids of taxane type.STE also provides a simple,component-safe,workable,and highly efficient method for the extraction of active natural product.

DNA extraction from fresh-frozen and formalin-fixed, paraffinembedded human brain tissue

Both fresh-frozen and formalin-fixed,paraffinembedded（FFPE）human brain tissues are invaluable resources for molecular genetic studies of central nervous system diseases,especially neurodegenerative disorders.To identify the optimal method for DNA extraction from human brain tissue,we compared methods on differently-processed tissues.Fragments of LRRK2 and MAPT（257 bp and 483 bp/245 bp）were amplified for evaluation.We found that for FFPE samples,the success rate of DNA extraction was greater when using a commercial kit than a laboratory-based method（successful DNA extraction from 76%versus 33%of samples）.PCR amplicon size and storage period were key factors influencing the success rate of DNA extraction from FFPE samples.In the fresh-frozen samples,the DNA extraction success rate was 100%using either a commercial kit（QIAamp DNA Micro）or a laboratorybased method（sample boiling in 0.1 mol/L NaOH,followed by proteinase K digestion,and then DNA extraction using Chelex-100）regardless of PCR amplicon length or tissue storage time.Although the present results demonstrate that PCR-amplifiable genomic DNA can be extracted from both fresh-frozen and FFPE samples,fresh brain tissue is recommended for DNA extraction in future neuropathological studies.

A New Homogenizing Technology to Obtain Rosmarinic Acid from Perilla Oil Meal

Objective To optimize the extraction technology of the active component, rosmarinic acid, an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid, in perilla oil meal for the first time by a new homogenizing technology called smashing tissue extraction (STE). Methods Orthogonal design was used to optimize the extraction condition. The content of rosmarinic acid was quantified from the methanol crude extract with the help of HPLC. Results The optimization of STE process to get rosmarinic acid from the perilla oil meal was the ratio of liquid to solid material at 10:1 and the power of extraction at 150 V, extracting twice (2 min for each time). Conclusion STE could be applied to extracting the active ingredients from the oil meals due to its high extraction efficiency. This new homogenizing technology has advantages on saving extraction time, raising extraction efficiency, and maintaining the temperature sensitive constituents.