Comparative anatomical and histological research of the roots of Rheum Palmatum L. And Rheum Officinale B.

Introduction. The aim of the study is to perform the anatomical and histological analysis of the roots of Rheum palmatum L. and Rheum officinale B., and also to check histochemical reactions for the presence of anthracene derivatives. Material and methods. The methodological basis of the anatomical and histological studies was the standard method for the preparation of micro-preparations of roots of Rheum palmatum L. and Rheum officinale B. described in the State Pharmacopoeia of the Russian Federation, XIV edition (OFS.1.5.3.003.15). Results and discussion. Anatomical and histological studies of Rheum palmatum L. and Rheum officinale B. raw materials confirmed the roots of Rheum palmatum L. to have classical secondary structure, while the roots of Rheum officinale B. have classical primary structure. The histochemical reaction to anthracene derivatives with 10% alkali solution was carried out, it resulted in cherry-red staining of core rays and some structures of cortical parenchyma of the fragment of Rheum palmatum L., while the roots of Rheum officinale B. failed to stain. Conclusions. The anatomical and histological studies of the rhizomes and roots of Rheum palmatum L. and Rheum officinale B. demonstrate the difference in the structure of closely related species of plants. It makes possible to identify the medicinal raw material of these plants.

An Optimized Protocol for Histochemical Detection of Senescence-associated Beta-galactosidase Activity in Cryopreserved Liver Tissue

Senescence-associated beta-galactosidase (SA-β-gal) activity assay is commonly used to evaluate the increased beta-galactosidase (β-gal) activity in senescent cells related to enhanced lysosomal activity. Although the optimal pH for β-gal is 4.0, this enzymatic activity has been most commonly investigated at a suboptimal pH by using histochemical reaction on fresh tissue material. In the current study, we optimized a SA-β-gal activity histochemistry protocol that can also be applied on cryopreserved hepatic tissue. This protocol was developed on livers obtained from control rats and after bile duct resection (BDR). A significant increase in β-gal liver activity was observed in BDR rats vs controls after 2 hr of staining at physiological pH 4.0 (6.98 ± 1.19% of stained/total area vs 0.38 ± 0.22; p<0.01) and after overnight staining at pH 5.8 (24.09 ± 6.88 vs 0.12 ± 0.08; p<0.01). Although we noticed that β-gal activity staining decreased with cryopreservation time (from 4 to 12 months of storage at −80C; p<0.05), the enhanced staining observed in BDR compared with controls remained detectable up to 12 months after cryopreservation ( p<0.01). In conclusion, we provide an optimized protocol for SA-β-gal activity histochemical detection at physiological pH 4.0 on long-term cryopreserved liver tissue:

Delivery of b-galactosidase gene in vivo with amphyphylic and combined liposomes

Цель исследования. Получение безопасного и эффективного вектора в целях генной терапии остается актуальной и нерешенной задачей, несмотря на усилия многих лабораторий в мире. Синтез веществ, имеющих сродство к ДНК и стабилизирующих ее спираль, а также обладающих группировками, опознаваемыми клетками нужных органов, является одним из перспективных направлений в таких исследованиях. Целью данной работы было изучение эффективности переноса в различные органы мышей репортерного гена бактериальной b-галактозидазы липосомами, содержащими синтетические холестериновые производные олигоэтиленпропиленимина (дихоленим и трихоленим), а также лактозилированный диглицерид (лактозолипид, LacS). Методика. Предварительно электронномикроскопически была подтверждена способность холенимов образовывать комплексы с ДНК оптимальных для трансфекции размеров (100-200 нм). Проверка холенимов на токсичность in vivo и введение липосом (фосфатидилхолин (ФХ)/дихоленим, ФХ/трихоленим и ФХ/лактозолипид/дихоленим в комплексе с плазмидой pCMV-SPORT-b-gal) в воротную вену печени проводились на самцах мышей линии ICR массой 30-40 г. Контролем служили интактные животные. Результаты. Cпособность холенимов образовывать комплексы с ДНК была подтверждена электронными микрофотографиями. Размеры комплексов (100-200 нм) оказались оптимальными для трансфекции in vivo , а холенимы - нетоксичными. Через 2 сут. после введения комплексов гистохимически на криосрезах внутренних органов животных с Х-Gal (субстрат) обнаружена отчетливо выраженная трансфекция органов, в том числе печени - главного объекта генной терапии. Заключение. Лактозилированный липид LacS и производные олигоэтиленпропиленимина холенимы являются безопасными, не менее и эффективными для достижения экспрессии функционального гена in vivo , чем коммерческие препараты, и перспективными для эффективной адресной доставки генов. The purpose of the work. Receipt of safe and effective vector for the purpose of genetic therapy remains the topical and unsolved problem inspite of many laboratories efforts in the world. Synthesis of substances which have affinity to DNA and stabilize its helix and also have the moieties recognized with the cells of the desired organs is one of the perspective ways in these investigations. Study of the effectivity of b-galactosidase bacterial reporter gene transfer with liposomes containing synthetic cholesteroyl derivatives of the oligoethylen/propylen/imine dicholenim and tricholenim and also lactosylated diglycerid (lactosolipid, LacS) to mouse organs was the purpose of this work. Methods. Previously ability of cholenims to form complexes with DNA of the optimal for transfection size (100-200 nm) was confirmed by electron microscopy technique. Testing the non-toxicity of cholenims in vivo and injections of phosphatidylcholine(Ph)/dicholenim, Ph/tricholenim and Ph/lactosolipid/dicholenim liposomes complexed with the pCMV-SPORT-b-gal plasmid to the liver portal vein were carried out on the mouse males of the ICR strain of 30-40 g of weight. The intact animals were as a control. Results. The ability of cholenims to form complexes with DNA was confirmed with electron micrographs. Complex sizes (100-200 nm) were optimal for in vivo transfection, and holenims were found non-toxic ones. The well-marked transfection of the animal’s inner organs including liver which is the main target of the genetic therapy was detected in two days after injection of the complexes by histochemical reaction of the cryosections with Х-Gal and the consequent light microscopy of the sections. Conclusion. It is concluded that the lactosylated lipid LacS and the oligoethylen/propylen/imine derivatives cholenims are not less safe and effective for achieving expression of the functional gene in vivo than the commercial ones and are promising for the effective targeted gene delivery.

THE ROLE OF ESTRADIOL IN THE SUSTENTATION OF FETOPLACENTAL SYSTEM

The aim of the study is to define the role of abnormalities in the formation of estriol hormones in the development of hormonal dysfunction of fetoplacental system. There were examined 39 placentas from women with exacerbation of chronic cytomegalovirus infection at the third trimester of pregnancy and IgG antibody titer 1:1600 and 30 placentas from women with latent CMV infection and titer 1:400. The activity of androstenedione dehydrogenase was found out with histochemical method on cryostat sections of freshly frozen tissues of placentas by Lloyd’s method. The assessment of intensiveness of histochemical reaction was done with cytophotometric method by Scion program. Estradiol in homogenate of placenta was measured with immune-enzyme method. The quantity of nuclei of placenta syncytiotrophoblast in the state of apoptosis was done with cytophotometric method on paraffin sections stained by ISEL method. By the results of the study the exacerbation of CMV infection at the third trimester of pregnancy leads to the decrease of intensiveness of histochemical reaction in placentas to androstenedione dehydrogenase till 32.1±2.88 pixel/mcm2 (at the latent course it is 54.2±4.31 pixel/mcm2, р&#60;0.001), and in homogenate of placenta to the decrease of estradiol till 18443.2±117.53 pmole/l (at the latent course of the disease it is 28977.7±158.13 pmole/l, p&#60;0.001), on paraffin sections in syncytiotrophoblast by the increase of nuclei number in the state of apoptosis till 5.0±0.03% (at latent course of the disease it is 1.2±0.001%, р&#60;0.001) and vacuole formation, which leads to destructive damages of syncytiotrophoblast cytoplasm. The obtained results prove that the exacerbation of CMV infection at the third trimester of pregnancy causes the decrease of estriol hormones formation, which is accompanied by the damage of morphostructure and exchange processes in cell elements of placenta.

INFLUENCE OF MAGNETOMICELLS BASED ON IRON NANOPARTICLES COATED BY CARBON ON STRUCTURE OF RAT LIVER

In this paper we study the effect of a single intravenous injection of suspension of magnitomitsell based on iron nanoparticles modified with carbon on the structure of the rat liver. Histological examination revealed the hemodynamic disturbances in the stroma of the organ and degenerative changes of hepatocytes from 1 to 40 days of the experiment. Perls histochemical reaction demonstrated the accumulation of modified iron nanoparticles in cells of the mononuclear phagocyte system of the liver. Number of Perlspositive macrophages decreased during the experiment from 1 to 40 per day.