Experimental Study On The Effect Of Focused Ultrasound Combined With Ultrasound Microbubble Loaded Mtx To Trophoblast

Focused ultrasound is used focused ultrasound energy source of anon-invasive technique in vitro, this technique is first put forward by Lynn,its development has experienced the process of more than50years. Focusedultrasound mainly through coupling with the skin, accurate positioning thetarget tissue, make the focus area of tissue temperature rise rapidly, within afew seconds to60℃or higher, so make the target tissue protein coagulation,owing to a lot of heat energy deposition damage, leading to coagulationnecrosis. At present, the focused ultrasound technique has been widely usedin clinical, however, more and more studies have found that the focusedultrasound application has certain limitation in recent years. First, when thetarget tissue is deep, the volume is big, rich blood supply, along with theincrease of the distance and the influence of blood flow, the energyattenuation is produced. In addition, the focused ultrasound treatment twodamage area will interfere with each other. In recent years, studies haveshown that ultrasound microbubble contrast agent can improve the curativeeffect of focused ultrasound effectively through change the soundenvironment targeted areas. Ultrasound microbubble loaded drugs have been popular research at home and abroad in recent years, and nanoscalemicrobubble has been produced. The research of focused ultrasoundcombined with ultrasound microbubble to clinical application of treatmentof solid tumors has been widely carried out.The trophoblast play an important role in the development of theplacenta. Embryos of trophoblast and tumor cells strikingly similarbiological characteristics and its by invading the uterus embryonic adhesionon the endometrium, then into vascular endothelial vascular reconstructionmatrix, thus the blood supply to the placenta, but unlike tumor infiltration oftrophoblast invasion is strictly control of time and space. By this regulation,trophoblast of human normal attack only1/3of the endometrium andshallow muscle layer. And placenta trophocyte abnormal invasion led to avariety of clinical disease, such as the placenta implantation, invasivehydatidiform mole, choriocarcinoma, placental site sertoli cell tumor, etc.In this experiment, we successfully prepared the ultrasoundmicrobubble loaded MTX, tested its properties, and discussed the effect ofpregnant rat placenta and the related mechanism by focused ultrasoundcombined with MTX ultrasonic micro bubble in vivo and in vitro experiment.This study to build a new type of drug dosage forms provides a new strategy,also for the treatment of trophoblast related diseases provides a new way ofthinking. This topic mainly includes the following three parts: PART I THE PREPARATION AND GENERAL PROPERTIESOF ULTRASOUND MTX LOADED METHOTREXATEObjective To prepare a kind of ultrasound microbubbles loadedMTX, study its physical properties, and evaluate as a new ultrasoundcontrast agent for drug delivery.Methods The MMTX were made using Double emulsion method. Itproperties were studied contained concentration, zeta potential, sizedistribution, drug entrapment efficiency and drug-loading amount. Primarystability experiments were performed including comparison of theproperties of ultrasound microbubbles loaded MTX under different storagecondition, before and after sterilizing with60Coγ irradiation. Drug releasedwith ultrasound was observed.Results MMTX had a concentration of approximately1.7×109～2.3×109／ml, Size distribution was368.7～619.6nm, a mean size of521.4nm, PM had a concentration of approximately3.4×109～4.1×109／ml,and drug entrapment efficiency was more than40%and drug-loadingamount was more than4.0%, zeta potential of-(8.2±6.65)mv. There was nosignificant difference in properties between before and after sterilizingwith60Coγ irradiation, properties were changed a little after being stored at4℃and-20℃for7days. Certain intensity of ultrasound energy canrupture microbubbles and release MTX. Conclusions In this study, we successfully prepared ultrasoundmicrobubbles loaded MTX and it was stable and the size waswell-distributed, this kind of microbubbles had high drag entrapmentefficiency, MTX could be released from microbubbles by ultrasoundirradiation.PART II EFFECT OF ULTRASOUND MICROBUBBLESLOADED MTX COMBINED WITHULTRASOUND-TARGETED MICROBUBBLE DESTRUCTIONON TROPHOBLAST OF HUMANObjective To study the cultivation parameters and suitable MTXtreatment concentration on HTR-8in vitro. To investigate the effects ofultrasound microbubbles loaded MTX combined with ultrasound-targetedmicrobubble destruction on cells ultrastructure, apoptosis, proliferation andexpression of apoptosis-related proteins and genes.Methods HTR-8cells were cultured in vitro, they growthcharacteristics were determined by cell counting and morphologicobservation. The suitable treatment concentration was decided according tothe proliferating inhibition rate of HTR-8cells in different dose groups，theproliferating inhibition rate was detected by CCK-8assay. HTR-8cellswere divided into6groups: blank control group(C), pure microbubblesand ultrasound group(PM+US), methotrexate group(MTX), microbubbles loaded methotrexate group(MMTX), methotrexate and ultrasoundgroup(MTX+US), microbubbles loaded methotrexate and ultrasoundgroup(MMTX+US). Proliferating inhibition rates at different time point ofcells were detected with CCK-8assay. Transwell invasion assay to detectthe invasiveness of HTR-8cells in vitro. Cell apoptosis was detected withAnnexin V-FITC technology. Protein and mRNA expression of Bcl-2, Bax,TIMP-1and MMP-9were separately detected by Western Blotting andreal-time PCR.Results During the course of cultivation, HTR-8cells wereinoculated with a concentration of1x105/m1. Certain concentration ofMTX could inhibit the growth of HTR-8cells, the inhibition effects werepositively related with the concentration of MTX and time, which showedan obvious dose-time-response relation. Compared with other groups, theproliferating inhibition effect of MMTX+US group was strongest and theapoptotic rate was highest (P<0.01), and the invasion of HTR-8cells wassignificantly lower than other groups (P <0.01). Many apoptosis bodieswere observed in MMTX+US group (P<0.01). The expression of TIMP-1and Bax in MMTX+US group was significantly higher than other groups atprotein and mRNA level, the Bcl-2expression in MMTX+US group waslowest among all groups (P<0.01).Conclusions MTX could inhibit the growth of HTR-8cells in vitro,1x109mol/L was chosen as the suitable concentration in following experiments. Microbubbles loaded MTX combined withultrasound-targeted microbubble destruction could distinctly inhibit theproliferation and induce apoptosis of HTR-8cells, the possible mechanismsof which may be lie in influencing the expression of apoptosis-relatedproteins and genes.PART III FOCUSED ULTRASOUND COMBINED WITHMICROBUBBLES LOADED MTX EFFECT ON PLACENTAOF PREGNANT RATObjective To explore the effect and relevant mechanism of focusedultrasound combined with microbubbles loaded MTX on placenta ofpregnant rat.Methods90pregnant rats were randomly divided into6groups:blank control group(C), pure microbubbles and ultrasoundgroup(PM+US), methotrexate group(MTX), microbubbles loadedmethotrexate group(MMTX), methotrexate and ultrasoundgroup(MTX+US), microbubbles loaded methotrexate and ultrasoundgroup(MMTX+US). To intervene in day19of pregnancy, the controlpregnant rat tail intravenous saline2ml, tail vein injection of MTXcontaining1mg medicine microbubble2ml in MMTX group, tail veininjection of2ml MTX in MTX group, the pregnant rats are open abdomenthen closed after exposure to10min in three groups. The rest of three groups of pregnant rats intravenous drug after30s used CZF irradiateplacenta, and the CZF was producted by Chongqing HIFU company, thenclosed abdomen after10min. Get out the placenta tissue after24h, proteinexpression of Caspase-3, Bcl-2, Bax, TIMP-1and MMP-9was detected byimmunohistochemistry(IHC), apoptosis was detected by TUNEL, mRNAexpression of Caspase-3, Bcl-2, Bax, TIMP-1and MMP-9were separatelydetected by real-time PCR.Results The expression of Caspase-3, Bcl-2, Bax, TIMP-1andMMP-9was found in all groups, the Bcl-2expression in MMTX+US groupwas lowest among all groups, the expression of Caspase-3, TIMP-1andBax in MMTX+US group was significantly higher than othergroups(P<0.01). Apoptotic index(AI) of MMTX+US group were highestamong all groups(P<0.01). The expression of Caspase-3, TIMP-1and Baxin MMTX+US group was significantly higher than other groups at proteinand mRNA level, the Bcl-2expression in MMTX+US group was lowestamong all groups (P<0.01).Conclusions Focused ultrasound and ultrasound microbubblesloaded MTX can increase apoptosis of the pregnant rat, and inhibtingplacenta cells infiltrating ability. The possible mechanisms of which maybe lie in influencing the expression of apoptosis-related proteins and genes.It provides the theory support and the new direction for placenta trophocytediseases treatment.